The Henryk Niewodnicza skiINSTITUTE OF NUCLEAR PHYSICS

Polish Academy of Sciences

152 Radzikowskiego str., 31-342 Kraków, Poland

www.ifj.edu.pl/reports/2009/

Kraków, December 2009

Report No. 2031/AP

XLII Polish Seminar on Nuclear Magnetic Resonance

and Its Applications. Kraków, 1-2 December 2009

ABSTRACTS

Organizing Committee:

Members:

T. Banasik Z.T. Lalowicz /v-chairman/

A. Birczy ski K. Majcher

J. Blicharski A. M ynarczyk

S. Heinze-Paluchowska M. Noga /secretary/

J. W. Hennel /chairman/ Z. Olejniczak

M. Jab o ska P. Rosicka

A. Jasi ski /honorary chairman/ W. Rutkowski

K. Jasi ski T. Skórka

A. Krzy ak G. Stoch

P. Kulinowski U. Tyrankiewicz

S. Kwieci ski W.P. W glarz /v-chairman/

G. Wo niak

Sponsors:

AMX-ARMAR AG,

BRUKER-Polska Sp. z o.o,

PA STWOWA AGENCJA ATOMISTYKI,

VARIAN INTERNATIONAL AG.

KOMITET FIZYKI POLSKIEJ AKADEMII NAUK

Addresses of the sponsors:

AMX-ARMAR AG

Anna Potrzebowska

ul. Bu garska 12a

93-362 ód

tel. (042) 645 00 64

BRUKER POLSKA SP. Z O.O

mgr W. Leszczy ski

ul. Budziszy ska 69

60-179 Pozna

tel. (061) 868 90 08

fax. (061) 868 90 96

e-mail: sekretariat@bruker.poznan.pl

www.bruker.pl

PA STWOWA AGENCJA ATOMISTYKI

ul. Krucza 36,

00-921 Warszawa

VARIAN NMR

mgr in . W. Ko mider

ul. Skarbka 21

60-348 Pozna

tel. (061) 867 31 84

tel. kom. 602 287 918

e-mail: woko@eranet.pl

www.varianinc.com

KOMITET FIZYKI POLSKIEJ AKADEMII NAUK

Wydzia III PAN

Instytut Fizyki PAN

Al. Lotników 32/46

02-668 Warszawa

CONTENTS

1. APPLICATION OF 19

F MAGNETIC RESONANCE IMAGING FOR THE STUDY

OF DRUGS EFFICACY EX VIVO.

Dorota Bartusik and Bogus aw Tomanek

2. TOCOTRIENOLS RELATED CHANGES IN HUMAN MCF-7

ADENOCARCINOMA USING 19

F MAGNETIC RESONANCE IMAGING EX

VIVO.

Dorota Bartusik, Boguslaw Tomanek, Danuta Siluk, and Roman Kaliszan

3. NON-CONTRAST BASED MRI OF HUMAN LUNG PERFUSION AND

VENTILATION.

Grzegorz Bauman, Michael Puderbach, Michael Deimling, Julien Dinkel, Christian

Hintze, Lothar R. Schad

4. CARBON-13 NMR RELAXATION STUDY OF THE INTERNAL DYNAMICS

IN CYCLODEXTRINS IN ISOTROPIC SOLUTION.

Piotr Bernatowicz, Katarzyna Ruszczy ska-Bartnik, Andrzej Ejchart, Helena

Dodziuk, Ewa Kaczorowska, and Haruhisa Ueda

5. STRUCTURAL CHANGES IN holo-S100A1 AND S100A1(C85M)PROTEIN.

CHEMICAL SHIFT BASED RESULTS.

Monika Budzi ska, Micha Nowakowski, Igor Zhukov, Agnieszka Belczyk, Andrzej

Bierzy ski, and Andrzej Ejchart

6. THE DISPERSION OF WATER PROTON SPIN-LATTICE RELAXATION RATES

IN AQUEOUS HUMAN PROTEIN HC ( 1-MICROGLOBULIN) SOLUTIONS.

Maria Dobies, Maciej Kozak, Stefan Jurga, and Anders Grubb

7. UNUSUAL STRUCTURE AND NMR SPECTRA OF SOME CYCLOPHANES.

Helena Dodziuk

8. INVESTIGATION OF TAUTOMERISM OF THE SELECTED OXOPURINES

IN NEUTRAL AND BASIC WATER SOLUTIONS BY 13

C NMR AND GIAO-DFT

CALCULATIONS.

Katarzyna Dybiec, Sergey Molchanov, and Adam Gryff-Keller

9. CEREBRAL WHITE MATTER RECOVERY IN ABSTINENT ALCOHOLICS –

A MULTIMODAL MAGNETIC RESONANCE STUDY.

Stefan Gazdzinski, Timothy C. Durazzo, Anderson Mon, Ping-Hong Yeh, and Dieter

J. Meyerhoff

10. INTERACTIVE EFFECTS OF AGE AND EXCESS BODY WEIGHT ON

NEURONAL VIABILITY - A MAGNETIC RESONANCE SPECTROSCOPY

STUDY.

Stefan Gazdzinski, Rachel Millin, Lana G. Kaiser, Susanne G. Mueller, Timothy C.

Durazzo, Michael W. Weiner, and Dieter J. Meyerhoff

1

2

3

4

5

6

7

8

9

11

11. TEMPERATURE INDUCED BOUND WATER IMMOBILIZATION IN

CETRARIA ACULEATA (SCHREB.) FR. THALLI OBSERVED BY PROTON

RELAXATION.

Hubert Hara czyk, P. Nowak, and M.A. Olech

12. MAPPING OF THE B1 FIELD USING ADIABATIC EXCITATION PULSES.

Franciszek Hennel

13. THE MULTIPARAMETRIC CMR-BASED ASSESSMENT OF HEART

FUNCTION IN MURINE MODEL OF HEART FAILURE TGaq*44.

Magdalena Jab o ska, UrszulaTyrankiewicz, Tomasz Skórka, Henryk Figiel, Sylwia

Heinze-Paluchowska, Miros aw Wo niak, and Stefan Ch opicki

14. MRI APPLICATION OF MICROSTRIP RF COILS.

Krzysztof Jasi ski, Peter Latta, Vyacheslav Volotovskyy, Anna M ynarczyk,

W adys aw P.W glarz, and Bogus aw Tomanek

15. TAUTOMERISM OF SCHIFF BASES DERIVED FROM (S)-

DIPHENYLVALINOL: THE SOLID STATE NMR AND X-RAY STRUCTURE

STUDIES.

Magdalena Jaworska, Pawe B. Hrynczyszyn, Katarzyna Nowicka, W odzimierz

Ciesielski, and Marek J. Potrzebowski

16. ADDUCTS FORMATION OF RHODIUM(II) TETRACARBOXYLATES WITH

METHIONINE DERIVATIVES: INVESTIGATIONS BY THE USE OF VIS AND

NMR TECHNIQUES.

Jaros aw Ja wi ski and Rafa G aszczka

17. MOLECULAR DYNAMICS AND STRUCTURE IN DIBLOCK COPOLYMERS

STUDIED BY NMR AND BROADBAND DIELECTRIC SPECTROSCOPY.

Jacek Jenczyk, Monika Makrocka-Rydzyk, Aleksandra Wypych, Stanis aw

G owinkowski, and Stefan Jurga

18. BACKBONE DYNAMICS OF TFE-INDUCED NATIVE-LIKE FOLD OF REGION

4 OF INITIALLY UNFOLDED E. coli RNA POLYMERASE 70

SUBUNIT.

Piotr Kaczka, Agnieszka Polkowska-Nowakowska, Krystyna Bolewska, Igor Zhukov,

Jaros aw Pozna ski, and Kazimierz L. Wierzchowski

19. fMRI QUALITY ASSURANCE SYSTEM: PHANTOM AND AUTOMATIC DATA

ANALYSIS.

Karolina Kami ska, Stanis aw Adaszewski, Ewa Pi tkowska-Janko, Piotr

Bogorodzki, and Maciej Pisklak

20. NMR RELAXATION MEASUREMENTS OF OXIDATION PROCESSES IN

BLOOD SERUM.

Joanna Kami ska, Jan Kobierski, and Barbara Blicharska

21. THE USEFULNESS OF DIFFUSION-WEIGHTED IMAGING IN

DISTINGUISHING BETWEEN SOME BRAIN PATHOLOGIES.

Al k d li fi d l k

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19

20

21

22

24

25

Aleksandra Klimas, Zofia Drzazga, and Ewa Kluczewska

22. CHEMICAL EXCHANGE PROCESSES IN HYDROGEN PEROXIDE

SOLUTIONS OBSERVED BY NMR RELAXATION.

Jan Kobierski, Hartwig Peemoeller, and Barbara Blicharska

23. DETECTOR FOR PROTON-ELECTRON DOUBLE RESONANCE IMAGING

(PEDRI).

ukasz Ko aszewski, Piotr Bogorodzki, Ewa Piatkowska-Janko, Jerzy Piotrowski,

Jerzy Skulski, and Maciej Pisklak

24. RECENT ADVANCES IN MULTIDIMENSIONAL NMR SPECTROSCOPY WITH

SPARSE RANDOM SAMPLING.

Wiktor Ko mi ski, Krzysztof Kazimierczuk, Maria Misiak, Jan Stanek, and Anna

Zawadzka-Kazimierczuk

25. THE ROLE of FULLEREN C60, CARBON NANOTUBES AND CARBON

ENCAPSULATED MAGNETIC NANOPARTICLES IN CREATING OF

CREATOL (5-HYDROXYCREATININE) AND UREMIC TOXIN -

METHYLGUANIDINE.

Hanna Krawczyk and Magdalena Pop awska

26. DO ESR AND NMR DESCRIBE THE SAME DYNAMICS OF PARAMAGNETIC

SYSTEMS?

Danuta Kruk

27. ANISOTROPIC DIFFUSION PHANTOM FOR B-MATRIX CALCULATION

Artur Tadeusz Krzy ak

28. ESR LINESHAPE FOR MULTISPIN SYSTEMS IN THE PRESENCE

OF LAXATION.

Aleksandra Kubica, Artur Mielczarek, and Danuta Kruk

29. ESTIMATION OF PHOSPHOLIPIDS CONCENTRATION IN PLASMA,

MONONUCLEAR CELLS, AND ERYTHROCYTES FROM PATIENTS WITH

HEMATOLOGICAL CANCERS - 31P MRS IN VITRO STUDY.

Ma gorzata Kuliszkiewicz-Janus

30. PERFORMANCE OF DFT, SOPPA, SOPPA(CCSD) AND CCSD(T) METHODS IN

PREDICTING NUCLEAR ISOTROPIC SHIELDINGS IN THE COMPLETE BASIS

SET LIMIT.

Teobald Kupka, Micha Stachów, Marzena Nieradka, Jakub Kaminsky, and Tadeusz

Pluta

31. THEORETICAL AND EXPERIMENTAL STUDIES ON CYTOSINE AND 5-

FLUOROCYTOSINE.

Teobald Kupka, Mariana Spulber, Mariana Pinteala, Adrian Fifere, Agnieszka

Raniszewska, and Malgorzata Broda

27

28

29

30

31

32

33

34

36

37

32. THEORETICAL CALCULATION OF NMR SHIELDINGS AND INDIRECT SPIN-

SPIN COUPLING CONSTANTS.

Teobald Kupka

33. CONFORMATIONAL ANALYSIS OF CYCLIC DYNORPHIN ANALOGUES

USING NMR DERIVED DATA.

Maria Kwasiborska, Agnieszka Zieleniak, Micha Nowakowski, Marta Oleszczuk,

Jacek Wójcik, Nga N. Chung, Peter W. Schiller, and Jan Izdebski

34. DYNAMICS OF HYDROXYL DEUTERONS AND BONDED WATER

MOLECULES IN NADY(0.8) ZEOLITE AS STUDIED BY MEANS OF

DEUTERON NMR SPECTROSCOPY.

Zdzis aw T. Lalowicz, Grzegorz Stoch, Artur Birczy ski, and M. Punkkinen

35. SPECTRAL DENSITY FUNCTIONS OF COMPLEX MOLECULAR MOTIONS.

Lidia Latanowicz and Zofia Gdaniec

36. SOLID STATE STUDIES OF CYCLODEXTRIN COMPLEXES WITH

ADAMANTANE AND AMANTADINE USING 13

C AND 15

N CP/MAS NMR.

Agnieszka Lis-Cieplak and Wac aw Ko odziejski

37. MOLECULAR DYNAMICS AND STRUCTURE OF MIKTOARM STAR BLOCK

COPOLYMERS BASED ON POLY(BUTYL ACRYLATE) AND

POLY(ETHYLENE OXIDE).

Monika Makrocka-Rydzyk, Aleksandra Wypych, Mariusz Jancelewicz, Stefan Jurga,

and Krzysztof Matyjaszewski

38. MOLECULAR DYNAMICS AND NUCLEAR RELAXATION PROCESSES OF

PLANAR CATIONS WITH PSEUDO 5-FOLD SYMMETRY AXES:

IMIDAZOLIUM AND PYRAZOLIUM COMPOUNDS.

W. Medycki, D. Kruk, R. Jakubas, and J. Jad yn

39. THEORY OF SOLID STATE DYNAMIC NUCLEAR POLARIZATION.

Artur Mielczarek, and Danuta Kruk

40. FIELD DEPENDENT RELAXATION PROCESSES IN MULTISPIN SYSTEMS.

Agnieszka Milewska and Danuta Kruk

41. THREE-DIMENSIONAL NMR SPECTROSCOPY OF ORGANIC MOLECULES.

Maria Misiak and Wiktor Ko mi ski

42. APPLICATION OF MR MICROSCOPY FOR ASSESSMENT OF HYDRATION

PROCESSES IN HPMC BASED MATRIX TABLETS.

Anna M ynarczyk, Marco L.H. Gruwel, Piotr Kulinowski, Krzysztof Jasi ski,

Przemys aw Doro y ski, Bogus aw Tomanek, and W adys aw P. W glarz

43. 3D NMR-BASED STRUCTURE OF apo-S100A1 HUMAN PROTEIN.

Micha Nowakowski, ukasz Jaremko, Igor Zhukov, Agnieszka Belczyk, Andrzej

Bierzy ski, and Andrzej Ejchart

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46

47

48

49

51

44. TOWARD CHIRAL CRYSTALS OF BENZODIAZACORONANDS.

Katarzyna Nowicka, Agata Jeziorna, Adam Sobczuk, Janusz Jurczak, Grzegorz D.

Bujacz, Anna Bujacz, W odzimierz Ciesielski, and Marek J. Potrzebowski

45. OPTIMIZATION METHOD FOR ADIABATIC TAGGING PULSES FOR

ARTERIAL SPIN LABELING.

Wojciech Obr bski, Piotr Bogorodzki, and Ewa Piatkowska-Janko

46. NON-DEBYE RELAXATION AND TEMPERATURE DEPENDENCE

OF THE SECOND MOMENT OF NMR LINE.

Marcin Olszewski and Nikolaj Sergeev

47. ANALYSIS OF BOLD SIGNAL IN THE BRAINSTEM DURING RESTING

STATE AND AFTER CONTINUOUS SOUND ACTIVATION.

Paulina Palowska, Michalina Ry , Uwe Klose, and Zofia Drzazga

48. SOLID STATE NMR STUDIES OF COORDINATION AND

ORGANOMETALLIC COMPLEXES OF NICKEL AND RUTHENIUM.

Piotr Paluch and Marek J. Potrzebowski

49.1H,

13C,

15N NMR COORDINATION SHIFTS IN CATIONIC Fe(II), Ru(II), Os(II)

WITH 2,2'-BIPYRIDINE AND 1,10-PHENANTHROLINE.

Leszek Pazderski, Tomasz Pawlak, Jerzy Sitkowski, Lech Kozerski, and Edward

Sz yk

50. SEEKING A MOLECULAR SWITCH - STUDY ON 7-HYDROXY -4-

METHYLQUINOLINE-8-CARBALDEHYDE IN SOLUTION.

Mariusz Pietrzak, Volha Vetokhina, Jacek Nowacki, Jerzy Herbich, and Andrzej L.

Sobolewski

51. SPIN-LATTICE RELAXATION IN DOPA-MELANIN AND MELANIN

ISOLATED FROM Sepia officinalis - COMPARATIVE EPR STUDIES.

Barbara Pilawa, Magdalena Zdybel, Daria Czy yk, Ewa Chodurek, and S awomir

Wilczy ski

52. SOLID-STATE CHARACTERIZATION OF S(+)CLOPIDOGREL

HYDROGENSULPHATE PHARMACEUTICALS AND THEIR POLYMORPHS.

Edyta Pindelska, Andrzej Mazurek, and Wac aw Ko odziejski

53. CONSTRUCTION OF HIGH FREQUENCY COILS FOR MRI AT 0.088 T.

Bartosz Proniewski, Henryk Figiel, and Tadeusz Pa asz

54. EPR STUDIES OF PARAMAGNETIC PROPERTIES OF THERMALLY

TREATED SISOMICIN AND VERAPAMIL.

Pawe Ramos, Barbara Pilawa, and Piotr Pepli ski

55. THE INFLUENCE OF TRACES OF WATER ON THE CHEMICAL SHIFTS

OF QULAENE IN BENZENE SOLUTION.

Micha Ra ew, Ewa Kaczorowska, Ewa Kula- wie ewska, Ewa Ciepicha , and Jacek

Wójcik

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58

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60

61

63

64

65

66

Wójcik

56. APPLICATION OF MRI FOR SPINAL CORD MONITORING IN ANIMAL

MODEL OF PRESSURE IMPACT INJURY.

Paulina Rosicka, Katarzyna Majcher, Wies aw Marcol, Wojciech lusarczyk, Tomasz

Banasik, Joanna Lewin-Kowalik, and W adys aw P. W glarz

57. INTERACTION OF SELECTED BICYCLIC MONOTERPENES WITH -

CYCLODEXTRIN. THERMODYNAMIC CHARACTERISTIC OF

COMPLEXATION.

Katarzyna Ruszczy ska-Bartnik, Micha Nowakowski, Helena Dodziuk, and Andrzej

Ejchart

58. SODIUM MR IMAGING.

Lothar R. Schad

59. THE CHANGES OF PHOSPHOLIPIDS IN LIPID RAFTS AT PATIENTS WITH

MYELODYSPLASTIC SYNDROME (MDS). .

Joanna Schiller, Ma gorzata Kuliszkiewicz-Janus, Izabela Dere -Wagamann, and

Stanis aw Baczy ski

60. RELAXATION PROCESSES OF SELECTED BIOMOLECULES.

Aleksandra Skowro ska and Danuta Kruk

61. IMPLEMENTATION OF RETROSPECTIVE GATING IN APPLICATION TO MR

ASSESSMENT OF CARDIAC FUNCTION IN MICE.

Tomasz Skórka, Sylwia Heinze-Paluchowska, Urszula Tyrankiewicz, and Magdalena

Jab o ska

62. INFLUENCE OF PARAMAGNETIC IONS PRESENCE ON RELAXATION IN

BLOOD SERUM.

Lech Skórski, Mateusz Synowiecki, and Barbara Blicharska

63. THE ITERATIVE APPROACH TO PROCESSING OF RANDOMLY SAMPLED

3D NMR SPECTRA.

Jan Stanek, and Wiktor Ko mi ski

64. STRUCTURE AND STEREOCHEMISTRY OF NTBC AND ITS METABOLITES -

A COMBINED NMR/DFT STUDY.

Przemys aw Szczeci ski, Anna Dziadecka and Adam Gryff-Keller

65. MOLECULAR DYNAMIC AND STRUCTURE OF PHOSPHATYDYLOCHOLINE

(DMPC)/CATIONIC GEMINI SURFACTANT (GEM-IK1) SYSTEM.

Kamil Szpotkowski, Aleksandra Wypych, Kosma Szutkowski, ad Maciej Kozak,

Stefan Jurga

66. MOLECULAR DYNAMICS IN PROTON CONDUCTING GEL/H3PO4

ELECTROLYTES STUDIED BY NMR.

L. Szutkowska, M. Dobies, K. Szutkowski, Stefan Jurga, G. ukowska, and

W. Wieczorek

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75

76

77

78

80

W. Wieczorek

67. METABOLIC PROFILE OF CEREBROSPINAL FLUID OBTAINED FROM

AMYOTHROPHIC LATERAL SCLEROSIS PATIENTS.

Beata Toczy owska

68. STRUCTURAL STUDIES OF N-TERMINAL SEQUENCE OF DERMORPHIN BY

MEANS OF SOLID STATE NMR SPECTROSCOPY AND XRD.

Katarzyna Trzeciak-Karlikowska, Agata Jeziorna, Grzegorz D. Bujacz, Anna Bujacz,

W odzimierz Ciesielski, and Marek J. Potrzebowski

69. NEW PERMANENT MAGNETS FOR MRI.

Krzysztof Turek, Piotr Liszkowski, and Boguslaw Tomanek

70. ASSESSMENT OF CARDIAC FUNCTION RESERVE IN MURINE MODELS OF

HEART FAILURE, IN VIVO STUDY.

U. Tyrankiewicz, T. Skórka, S. Heinze-Paluchowska, M. Jab o ska, M. Wozniak, L.

Drelicharz, and S. Chlopicki

71. LOCAL AND COOPERATIVE DYNAMICS IN MODIFIED

POLY(DIMETHYLSILOXANE)S STUDIED BY FAST FIELD CYCLING NMR,

BROADBAND DIELECTRIC AND FOURIER TRANSFORM INFRARED

SPECTROSCOPIES.

Wiktor Waszkowiak, Aleksandra Wypych, Maria Dobies, Stefan Jurga, and Hieronim

Maciejewski

72. A NOVEL DETERMINATION OF 13

C NUCLEAR MAGNETIC MOMENT

IN TERMS OF THE 3He FROM GASEOUS

13CH4 NMR SPECTRA

Marcin Wilczek , Anna Szyprowska, and W odzimierz Makulsk

73. EPR EXAMINATION OF FREE RADICALS IN RADIATIVE STERILIZED

PIPERACILLIN.

S awomir Wilczy ski, Barbara Pilawa, Marta Ptaszkiewicz, Janusz Swako , Pawe

Olko, Robert Koprowski, and Zygmunt Wróbel

74. FREE RADICALS PROPERTIES OF DIETARY SUPPLEMENTS BASED ON

FISH AND PLANT OILS APPLIED IN COSMETOLOGY.

Wilczy ski S awomir, Zdybel Magdalena, Barbara Pilawa, Deda Anna, and

Pierzcha a Ewa

75. RELAXATION PARAMETERS OF GD(III) AND MN(II) IONS IN WATER FROM

THE PERSPECTIVE OF CONTRAST AGENTS.

Milosz Wojciechowski and Danuta Kruk

76. UNIPLANAR GRADIENT COILS - FROM DESIGN TO APPLICATIONS.

Grzegorz Wo niak, Tomasz Skórka, Krzysztof Jasi ski, Tomasz Banasik,

Mohammad Mohammadzadeh, Dominik von Elverfeldt, Jürgen Hennig, and

W adys aw P. W glarz

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77. DYNAMICS OF POLYSTYRENE OF DIFFERENT MOLECULAR

ARCHITECTURE AS REVEALED BY NUCLEAR MAGNETIC RESONANCE,

BROADBAND DIELECTRIC SPECTROSCOPY AND REOLOGY.

Aleksandra Wypych, Monika Makrocka-Rydzyk, Grzegorz Nowaczyk, Eugeniusz

Szcze niak, Stefan Jurga, Akira Hirao, and Takumi Watanabe

78. SPECTRA OF HIGH DIMENSIONALITY FOR EASY RESONANCE

ASSIGNMENT IN PROTEINS.

Anna Zawadzka-Kazimierczuk, Krzysztof Kazimierczuk, and Wiktor Ko mi ski

79. EFFECT OF TEMPERATURE ON EPR SPECTRA OF DOPA-MELANIN-

NETILMICIN COMPLEXES.

Magdalena Zdybel, Barbara Pilawa, Ewa Buszman, Dorota Wrze niok, Ryszard

Krzyminiewski, Zdzis aw Kruczy ski, Robert Koprowski, and Zygmunt Wróbel

80. SPIN-LATTICE RELAXATION IN THERMALLY STERILIZED

CHLORTALIDONE.

Magdalena Zdybel, Adamczyk, Barbara Pilawa, Magdalena Ko cielniak, and Daria

Czy yk

81. DISPERSION MEASUREMENTS OF T1 IN PROTEIN SOLUTIONS.

ukasz elazny, Dorota Wierzuchowska, and Barbara Blicharska

82. THE ONE NMRTM

PROBE FROM VARIAN AND THEIR COMBINATION

TOOLS.

Thomas Zellhofer

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APPLICATION OF 19F MAGNETIC RESONANCE IMAGING FOR THE STUDY OF DRUGS EFFICACY EX VIVO

Dorota Bartusik1 and Bogusław Tomanek1,2,3,4

1National Research Council Canada, Institute for Biodiagnostics (West), Calgary, Alberta, Canada; 2Cross Cancer Institute, Department of Medical Physics, Edmonton, Alberta, Canada; 3Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland;

4University of Alberta, Department of Oncology, Edmonton, Alberta, Canada

Oncology is the major beneficiary of pre-clinical and clinical applications of 19F NMR. 19F nuclei is used in pharmaceutical investigations of anti-cancer drugs because 19F stabilizes drugs and is responsible for interactions with intracellular microenvironment, thus drugs efficacy.

The aim of our study was to apply 19F Magnetic Resonance Imaging at 9.4 T (Cross Cancer Institute, Edmonton, Canada) to observe drug efficacy. We labeled Herceptin (Trastuzumab, Genentech Inc., San Francisco, CA) with fluorine in the form of perfluorocarbon (PFCE, perfluoro-15-crown-5-ether). For the study we selected human breast cancer cell line MCF-7 with stable positive over-expression of HER-2 protein. On the cell surface HER-2 is recognized as Herceptin receptor. As control we used Human mammary epithelial cells (HMEC). The three dimensional cell cultures were established using Hollow Fiber Bioreactor (HFB, FiberCell System Inc., Frederick, MD). 19F MRI was used for visualization of the cellular uptake of new fluorine labeled Herceptin.

We observed that the oil-water emulsion of Herceptin with PFCE was more efficient than Herceptin alone in MCF-7 culture. Normal (HMEC) cells did not respond to any treatment. A significant correlation between duration of treatments and MCF-7 cells viability was observed. 19F signal intensity increased due to 19F uptake, however the cells that were successfully treated were no longer possible for viability assays with trypan blue. The use of HFB device allowed high-density 3-D cell cultures in the reproducible experimental setup and provided controlled conditions during biochemical and MR study.

1

TOCOTRIENOLS RELATED CHANGES IN HUMAN MCF-7 ADENOCARCINOMA USING 19F MAGNETIC RESONANCE

IMAGING EX VIVO

Dorota Bartusik 1, Boguslaw Tomanek 1,2,3,4, Danuta Siluk 5 , and Roman Kaliszan 5

1National Research Council Canada, Institute for Biodiagnostics (West), Calgary, Alberta,

Canada; 2Cross Cancer Institute, Department of Medical Physics, Edmonton, Alberta, Canada; 3University of Alberta, Department of Oncology, Edmonton, Alberta, Canada; 4Polish Academy of Sciences, Institute for Nuclear Physics, Kraków, Poland; 5Medical

University of Gdańsk, Department of Biopharmaceutics and Pharmacodynamics, Gdańsk, Poland

Tocotrienols, analogues of vitamin E, are significant sources of antioxidant activity to

all living cells. Cell viability is related to oxygen content and can be used to study the growth of breast cancer cells. Therefore we study the oxygen content using MR methods to measure tocotrienols’ efficacy.

In the present study we used 19F Magnetic Resonance Imaging (MRI) measurements for assessing oxitometry of MCF-7 cells after α-, γ-, δ- tocotrienols treatments. The cells were grown in three dimensional (3-D) cultures using MR compatible Hollow Fiber Bioreactor (HFBR) device. We studied the changes in the intracellular oxygen concentration in 3-D cultures caused by hexafluorobenzene (HFB) uptake in control and treated cells. The 19F spin–lattice relaxation of HFB is highly sensitive to oxygen concentration and minimally to temperature that is useful to measure intracellular oxygen concentrations. The cells’ viability was assayed using trypan blue.

The cells were treated with tocotrienols (0-1000 µM) during 72 h. The significant viability changes were observed for concentrations higher than 250 µM of α-tocotrienol, 200 µM of γ-tocotrienol and 150 µM of δ- tocotrienol, as compared to control. The viability decreased from 93 ± 4% to 52 ± 4%, 45 ± 9% and 38 ± 6%, respectively. The γ- and δ- tocotrienols were the most inhibitory compounds concerning effects of tocotrienols on MCF-7 cells’ growth. The IC50 values for α-, γ-, and δ-tocotrienols was 14 µM, 19 µM and 7 µM, respectively. The oxygen concentrations in the cultures were 13.0 %O2, 11.0 %O2, 9.0 %O2, respectively. Significant correlation between the oxygen and vitamins concentrations, that was related to MCF-7 cells’ viability, was observed.

2

NON-CONTRAST BASED MRI OF HUMAN LUNG PERFUSION AND VENTILATION

Grzegorz Bauman1,4, Michael Puderbach2, Michael Deimling3, Julien Dinkel2, Christian Hintze2, and Lothar R. Schad4

1German Cancer Research Center, Medical Physics in Radiology, Heidelberg, Germany; 2German Cancer Research Center, Department of Radiology, Heidelberg, Germany;

3Siemens Healthcare, Erlangen, Germany; 4Computer Assisted Clinical Medicine, University of Heidelberg, Mannheim, Germany

Assessment of regional pulmonary perfusion and ventilation has important clinical value as an indicator of lung function. Current standard MR based methods for evaluation of lung perfusion and ventilation are dependent on the application of the inhalative or the intravenous contrast agents. We propose a Fourier Decomposition method for non-contrast-enhanced functional lung MRI [1]. The method was adapted on a 1.5 T clinical MR scanner using fast acquisition and submillisecond echo sampling with a Steady-State Free Precession (SSFP) sequence to produce time-resolved 2D data stacks [2]. All measurements were performed on a 1.5 whole-body MR scanner. Seventeen healthy volunteers (11 men, 6 women) with mean age of 36.5 ± 14.1 years (age range: 19 - 64 years) were investigated. Set of five coronal slices (S1-S5) was acquired in every subject using time-resolved scans with a 2D+t SSFP sequence (TR/TE = 1.9 ms/0.8 ms and TA = 116 ms/image) to cover the chest volume. Each time-resolved set consisted of n = 198 images for every slice location with a total acquisition time T = 59.4 s, spectral resolution ∆f = 1/T = 0.017 Hz and spectral width fB = 1/(2·TA) = 1.667 Hz. The identical measurement protocol was repeated after 24 hours. Neither breath-holding nor ECG triggering were required. Images in every data stack containing single slice were corrected for the respiratory motion using fully automatic non-rigid registration algorithm [3]. Pixel-wise application of Fourier Transform along the time axis of the data stack converted them into sets of n images representing spectral frequencies fn ∈0, ∆f, ... , fB. After the periodical signal intensity changes with regard to the cardiac and respiratory cycles were identified on the frequency spectra, integration of appropriate spectral ranges produced perfusion- and ventilation-weighted images. The images showed a homogenous signal distribution without defects for every healthy volunteer. Medical and technical reproducibility of the method was examined. The presented method requires only minimal patient compliance and is not dependent on triggering techniques. Neither administration of intravenous contrast agents nor inhalative gaseous media like hyperpolarized helium [4], oxygen [5] is needed. Further studies are required to determine the clinical impact of the proposed method.

References: 1. Deimling M. et al. Proc. 16th annual meeting ISMRM, 2008, Toronto, Canada, p. 2639; 2. Bauman G. et al. Magn Reson Med, 2009 Sep;62(3):656-64; 3. Chefd’hotel C. et al. VLSM'2001, ICCV Workshop, 2001, Vancouver, Canada; 4. De Lange E.E. et al. Radiology, 1999; 210: 851-857; 5. Edelman R.R. et al. Nat. Med., 1999; 2: 1236-1239.

3

CARBON-13 NMR RELAXATION STUDY OF THE INTERNAL DYNAMICS IN

CYCLODEXTRINS IN ISOTROPIC SOLUTION

Piotr Bernatowicza, Katarzyna Ruszczyńska-Bartnikb, Andrzej Ejchartb, Helena Dodziuka, Ewa Kaczorowskab, and Haruhisa Uedac

aInstitute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland;

bInstitute of Biochemistry and Biophysics, Warsaw, Poland; cDepartment of Physical Chemistry, Hoshi University, Tokyo, Japan

13C nuclear spin relaxation processes in 6- to 12-membered cyclodextrins (from

α to η) were investigated in D2O solution at multiple magnetic fields. Detailed analysis of the 13C longitudinal relaxation, the 13C relaxation in rotating frame and the 1H-13C nuclear Overhauser enhancement (NOE) in these molecules yielded their rotational diffusion tensors and semiquantitative picture of the internal motions these oligosugars undergo. The dynamics in α- and β- molecules seems to be different than the motional behaviour of their higher analogues. The results suggest that on the time scale of molecular tumbling none of the investigated cyclodextrins takes rigid truncated-cone conformation.

4

STRUCTURAL CHANGES IN holo-S100A1 AND S100A1(C85M)PROTEIN.

CHEMICAL SHIFT BASED RESULTS

Monika Budzińska1), Michał Nowakowski1), Igor Zhukov1’2), Agnieszka Belczyk1), Andrzej Bierzyński1), and Andrzej Ejchart1)

1)Institute of Biochemistry and Biophysic, Polish Academy of Sciences, Warszawa Poland;

2)Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia S100A1 is a homodimeric calcium binding protein. Each subunit, built up of 93 residues, contains two EF-hand motifs linked with a flexible linker [1]. The EF-hand motif is composed of a sequence helix –loop – helix. The protein undergoes a conformational change upon binding calcium in order to interact with protein targets and initiate a biological response [2]. Possible structural changes among apo-S100A1, holo-S100A1 and S100A1(C85M) proteins can be detected using ∆δtot parameter based on the chemical shift data [3]: ∆δtot=( ∆δHN)2 + 0.024(∆δN)2 + 0.116(∆δCO)2 + 0.076(∆δ Cα)21/2

On the other hand, secondary structure elements can be predicted via the evaluation of Φ and Ψ backbone torsion angles using programs: PREDITOR [4] and TALOS [5] which are essentially based on the backbone chemical shifts as well. Using double labeled 15N/13C apo-S100A1 and holo-S100A1 proteins we have assigned the chemical shifts of almost all backbone nuclei in both proteins. Protein secondary structure elements have been determined and compared pointing out to distinctive conformational changes due to the modification of crucial C85 residue or calcium binding. Acknowledgment: This work was supported by a grant N301 031234 from the Polish Ministry of Science and Higher Education References: [1] R. Donato, Int. J. Biochem. Cell Biol. (2001)33, 637-668. [2] N.T. Wright, K.M. Varney, K.C. Ellis, J. Markowitz, R.K. Gitti, D.B. Zimmer,

D.J. Weber, J. Biol. Mol. (2005)353, 410-426. [3] Ayed, F.A.A. Muller, G.S. Yi, Y. Lu, L.E. Kay, C.H. Arrowsmith, Nature Struct. Biol.

(2001)8, 756-760. [4] M.V. Berjanskii, S. Neal, D.S. Wishart, Nucleic Acids Res. 2006 Jul 1;34 [5] G. Cornilescu, F. Delaglio, A. Bax, J. Biomol. NMR (1999) 13, 289-302.

5

THE DISPERSION OF WATER PROTON SPIN-LATTICE RELAXATION RATES IN AQUEOUS HUMAN PROTEIN HC

(α1-MICROGLOBULIN) SOLUTIONS

Maria Dobies1, Maciej Kozak1, Stefan Jurga1, and Anders Grubb2

1Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University in Poznań, Poznań, Poland; 2Department of Clinical Chemistry,

University Hospital, Lund, Sweden

The human protein HC (α1 – microglobulin) is a low molecular weight heterogeneous glycoprotein widely distributed in the human body fluids and belonging to the lipocalin superfamily. This biomolecule is produced by the human liver and used in the clinical routine as a sensitive indicator of tubular dysfunction. The molecular mass of the glycosylated protein is about 27 kDa. The monomer of protein HC is characterized by a radius of gyration RG=2.16 nm and the dimer by RG=2.93 nm [1].

The 1H NMR Fast Field Cycling relaxometry was applied to study the molecular dynamics of the human protein HC (α1-microglobulin), its hydration and aggregation in solution state. 1H NMRD data for HC protein solutions at three different concentrations: 0.28 mM, 0.56 mM and 0.83 mM are collected in figure 1.

Figure 1. 1H NMRD data of human protein HC in water solution at different concentrations (0.28 mM, 0.56 mM and 0.83 mM). The best fit model-free profiles (n=3) and best fit Lorentzian profiles are assigned by the solid and dotted lines, respectively.

All 1H NMRD profiles exhibit strong stretching towards low Larmor frequency and have a two-step shape. The 1H NMRD data have revealed the complex nature of the water/protein HC system resulting from the co-existence of monomer and dimer forms of the protein in solution as well as the presence of oligosaccharides linked to the polypeptide chain. A comparison of the average correlation time values <τc> obtained from the model-free [2] fits with the values predicted on the basis of hydrodynamic τr theory, suggests that the dynamics in solution state is governed mainly by the dimer form of the protein HC (the dominant contribution to the water proton-spin lattice relaxation comes from exchanging protons from the surface of the dimer). The existence of small number of oligomeric forms of the protein HC in solutions is postulated because of the two-step shape of water proton spin-lattice relaxation rate dispersion profiles [3]. References: [1] Kozak M., Grubb A., Protein&Peptide Letters, 14 (2007) 425 [2] Halle, B.; Jóhannesson, H.; Venu, K. Journal of Magnetic Resonance. 135 (1998) 1 [3] Dobies M., Kozak M., Jurga S., Grubb A., Protein&Peptide Letters, 16 (2009) 1496

6

UNUSUAL STRUCTURE AND NMR SPECTRA OF SOME CYCLOPHANES

Helena Dodziuk

Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Po

Cyclophanes with small bridges, like known 10 exhibiting a distorted ring structure are excitinmeasurements and the calculations of chemical shto X-ray analysis, a unique opportunity to mongeometry. Therefore, combined measurements anbasic systems as para-[2.2]cyclophane 1 and [2.2.2]cyclophanes with three bridges 6 – 9 and pSome calculations have been also carried out cyclophanes 3 NMR spectra of which has beencalculations of chemical shifts and coupling conpublished while practically no calculations for NMour paper7 on 6 – 9.

The results obtained indicate that the comcomputation of chemical shifts and coupling consstrained hydrocarbons. In particular, the literatucompound has not been sufficiently pure.

References: (1) Modern Cyclophane Chemistry; R. Gleiter, H. Hopf, Ed(2) Hopf, H. In Strained hydrocarbons. Beyond van't Hoff a

Weinheim, 2009; (3) H. Dodziuk, M. Ostrowski, Eur. J. Org. Chem., 2006, 5(4) Lin, S.-T.; Yang, F.-M.; Liang, D. W. J. Chem. Soc. Per(5) L. Ernst, Ann. Rep. NMR Spectroscopy 2006, 60, 77; (6) L. Ernst, Progr. Nucl. Magn. Res. 2000, 37, 47; (7) H. Dodziuk, M. Ostrowski, K. Ruud, J. Jaźwiński, H. H

407.

H H

H

H

1 2 3

6 7 8

7

land

1 – 4, 6 - 9 and some hypothetical ones, like g objects of stereochemical studies1,2. NMR ifts and coupling constants offer, in addition itor structural distortions from a standard d calculations of NMR parameters of such its derivatives, superphanes 4, 53, some

ara-[3.3]cyclophane 10 seem of importance. for cis- 2 and trans-hexahydro[2.2]para-

published4. It should be stressed that few stants for strained hydrocarbons have been R parameters of cyclophanes exist5,6 except

bination of NMR measurements and the tants is a powerful tool in studies of highly re data for 34 seem to indicate that this

s., Wiley-VCH, Weinheim, 2004; nd LeBel hypothesis; H. Dodziuk, Ed. Wiley-VCH,

231; kin 1 1999, 1725;

opf, W. Koźmiński, Magn. Res. Chem., 2009, 47,

4 5

9 10a 10b

INVESTIGATION OF TAUTOMERISM OF THE SELECTED OXOPURINES IN NEUTRAL

AND BASIC WATER SOLUTIONS BY 13C NMR AND GIAO-DFT CALCULATIONS

Katarzyna Dybiec, Sergey Molchanov, and Adam Gryff-Keller

Faculty of Chemistry, Warsaw University of Technology

Warsaw, Poland

2-Oxopurine, 6-oxopurine (hipoxanthine), 8-oxopurine and 2,6-dioxopurine (xanthine) exist in neutral or basic water solutions as neutral molecules, monoanions and/or dianions (Fig. 1). Moreover, most of them are equilibrium mixtures of tautomers undergoing fast mutual transformations.

N

N NH

HN

8OP-CO-N7H-N9H

OHN

N NH

N

2OP-CO-N1H-N9H

O

HN

N N

HN

O

Hyp-CO-N1H-N7H

HN

NH

N

HN

O

O

Xan-CO-N1H-N3H-N7H Figure 1. Investigated compounds as represented by the most abundant tautomers of

neutral molecules: 2-oxopurine (2OP), hypoxanthine (Hyp), 8-oxopurine (8OP) and xanthine (Xan).

In order to determine the structures of the species present in water solutions and to

estimate their populations, the series of 13C NMR spectra of the investigated compounds in solutions of various acidities have been recorded. The analysis of the δ(pH) dependences allowed the spectra of particular forms of a given compound to be retrieved. Then, the 13C chemical shifts determined in that way were expressed as the population-weighted sums of the shielding constants calculated theoretically for the tautomers which were expected to be the most abundant species in the investigated solutions. Such a two-step analysis allowed the choice of the important structures to be verified and their populations to be estimated.

The molecular structure optimizations and calculations of shielding constants were performed for oxopurine species solvated by three hydrogen-bonded water molecules using the DFT and GIAO-DFT methods with B3LYP functional, 6-311++G(2d,p) basis set and polarizable continuum model (PCM) for including the impact of the bulk solvent.

8

CEREBRAL WHITE MATTER RECOVERY IN ABSTINENT ALCOHOLICS – A MULTIMODAL MAGNETIC RESONANCE STUDY

Stefan Gazdzinski, Timothy C. Durazzo, Anderson Mon, Ping-Hong Yeh, and Dieter J. Meyerhoff

Center for Imaging of Neurodegenerative Diseases, DVA Medical Center, and University of California, San Francisco, California

Introduction: Most previous neuroimaging studies of alcohol-induced brain injury and recovery thereof during abstinence from alcohol used a single imaging modality. They demonstrated widespread microstructural, macrostructural, or metabolite abnormalities that were partially reversible with abstinence [1,2], with cigarette smoking potentially modulating these processes [3]. The goals of this study were to evaluate white matter (WM) injury and recovery thereof simultaneously with diffusion tensor imaging (DTI), magnetic resonance imaging (MRI) and spectroscopy in the same cohort and to evaluate relationships between outcome measures of similar regions.

Methods: Sixteen non-smoking alcohol-dependents (nsALC) and 20 smoking individuals (sALC) were scanned at 1.5 Tesla, at approximately one week of abstinence from alcohol. Ten nsALC and 11 sALC were rescanned at approximately one month of abstinence. 22 non-smoking light-drinkers were also scanned only once. Structural and spectroscopy data were acquired with T1-weighted (TR/TI/TE=10/300/4 ms), and multislice 1H MRSI (TR/TI/TE=1800/300/25 ms) sequences, respectively. The latter was obtained in 3 parallel planes through the centrum semiovale, nuclei of the basal ganglia, and cerebellum. Diffusion weighted images were acquired with a single-shot EPI sequence (TR/TE/TI=5000/100/3000ms, 2.4x2.4x5mm3) with a double refocusing SE and bipolar external diffusion gradients [4] to minimize eddy-current artifacts without sacrificing SNR. Six encoding directions and five b-values (0,160, 360, 640, and 1000 sec/mm2) were used. The T1-weighted images were segmented into gray matter, white matter (WM), and cerebrospinal fluid (CSF) of major lobes with automated probabilistic segmentation, aided by an automated atlas-based region labeling of major lobes, cerebellum, and subcortical structures. Regional atrophy-corrected metabolite concentrations of N-acetyl-aspartate (NAA, a marker of neuronal viability), choline-containing compounds (Cho), myo-inositol (m-Ino) and creatine containing metabolites (Cr), were calculated by combining spectroscopic and segmented MRI data. For DTI analyses, median FA and MD in frontal, parietal, temporal, and occipital WM were calculated using only diffusion voxels with FA>0.2 and WM>95%.

Results: At one week of abstinence, nsALC had higher MD in frontal, temporal, and parietal WM (all p<0.008) relative to nsLD, while sALC had elevated MD only in frontal WM (p=0.03) relative to nsLD. Even in the absence of significant atrophy, nsALC and sALC each demonstrated lower concentrations of NAA in frontal and parietal WM compared to nsLD (both p=0.01). Over one month of abstinence, MD decreased in all regions of nsALC (p<0.04), while FA increased only in temporal WM (p=0.003). No corresponding changes were observed in sALC. By contrast, WM volume in sALC increased over one month of abstinence in all WM regions (p<0.04), whereas there were no changes in nsALC. The longitudinal changes in concentrations of NAA and Cho were not significant in this sample.

Discussion: These data demonstrate significant WM changes with abstinence from alcohol, reflected either as microstructural recovery or volumetric increases depending on the smoking status of the participants. The changes in WM were not accompanied by changes in NAA, suggesting that these changes were driven by the glial component of WM. In conclusion, our results are important, as they demonstrate that 1) not controlling for smoking status may lead to varying results depending on the proportion of smokers in the cohort and 2) use of a single imaging

9

modality provides an incomplete picture of neurobiological processes associated with alcohol induced brain injury and recovery thereof.

References: 1. Pfefferbaum, A., et al., Neurobiol Aging, 2006. 27(7): p. 994-1009. 2. Sullivan, E.V., NIAAA Research Monograph No. 34:NIAAA, 2000, Bethesda, MD. p. 473-508. 3. Durazzo, T.C., et al., Alcohol Alcohol, 2007. 42(3): p. 174-85. 4. Reese, T.G., et al., Magn Reson Med, 2003. 49(1): p. 177-82.

10

INTERACTIVE EFFECTS OF AGE AND EXCESS BODY WEIGHT ON NEURONAL VIABILITY – A MAGNETIC RESONANCE

SPECTROSCOPY STUDY Stefan Gazdzinski, Rachel Millin, Lana G. Kaiser, Susanne G. Mueller, Timothy C. Durazzo,

Michael W. Weiner, and Dieter J. Meyerhoff

DVA Medical Center, University of California San Francisco, San Francisco, USA Background: Excessive body weight is associated with brain structural alterations, poorer cognitive function [1], and lower prefrontal glucose metabolism [2], and reports suggest that excessive body weight accelerates aging processes [3]. We demonstrated widespread decreases in concentrations of N-acetyl-aspartate (NAA, a marker of neuronal viability, associated with glucose utilization) in a healthy middle-aged cohort, especially in frontal lobe [4], and in anterior cingulate cortex (part of frontal lobe) of healthy elderly [5] as a function of higher body mass index (BMI; a measure of body fat). As NAA was found to depend on age in cohorts with age range spanning several decades (e.g., [6]), we hypothesized that elevated BMI is associated with faster NAA decline with advancing age. Methods: We used data from 46 healthy, highly functioning participants (55.3 ± 19.6 years; range: 22-84years; BMI = 25.0 ± 3.0 kg/m2; range: 19.0-33.9kg/m2) from studies on brain aging and Gulf War syndrome. Proton magnetic resonance spectroscopy (1H MRS) at 4 Tesla measured concentrations of NAA, glutamate (Glu, involved in cellular metabolism), choline-containing compounds (Cho, involved in membrane metabolism), and creatine (Cr, involved in high energy metabolism) in anterior (ACC) and posterior cingulate cortices (PCC). As absolute metabolite concentrations were not available, we scaled metabolite concentration to Cr. Individual ratios were modeled as a function of age, BMI, and interactions between age and BMI.

Figure: Illustration of the trend for statistical interaction between age and BMI: the inverse association between BMI and NAA/Cr is stronger among participants older than 50 years compared to their younger counterparts. Results: Neither BMI nor age were significant predictors of NAA/Cr in ACC; however, there was a trend for an interaction between BMI and age (p=0.054), reflecting stronger association

BMI [kg/m2]

18 20 22 24 26 28 30 32 34

NAA

/Cr i

n A

CC

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7Age > 50 years

BMI [kg/m2]

18 20 22 24 26 28 30 32 34

NA

A/C

r in

AC

C

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7Age < 50 years

11

between higher BMI and lower NAA/Cr in older participants than in their younger counterparts (illustrated in the Figure below); this result can be interpreted as faster NAA/Cr decline among participant with higher BMI as well. Advancing age (p=0.005), but not BMI or the interaction between age and BMI, was associated with decreasing NAA/Cr in PCC. Conclusions: Our results suggest that elevated BMI is associated with faster decline in neuronal viability with age only in ACC. Conversely, BMI was not associated with NAA/Cr in PCC; the NAA/Cr decline in PCC depended solely on age. Taken together, the adverse effects of excess body weight on neuronal viability appear to accumulate over a lifetime. References: 1. Beydoun MA, et al., Obes Rev 2008;9(3):204-18. 2. Volkow, N.D., et al, Obesity: doi: 10.1038/oby.2008.469. 3. Valdez et al., Lancet 2005; 366: 662–64. 4. Gazdzinski, S., et al., Ann Neurol, 2008. 63(5): p. 652-7. 5. Gazdzinski S., et al., Obesity, in Press 6. Schuff N, et al., Neurobiol Aging 1999;20:279-285.

12

TEMPERATURE INDUCED BOUND WATER IMMOBILIZATION IN CETRARIA ACULEATA (SCHREB.) FR. THALLI OBSERVED

BY PROTON RELAXATION

Hubert Harańczyk1, P. Nowak1, and M.A. Olech2

1Institute of Physics and 2Institute of Botany, Jagiellonian University, Cracow, Poland

Numerous Antarctic lichen species may resist low temperatures in their habitat and may dehydrate to extremely low hydration level and rehydrate from gaseous phase to the hydration level sufficient to initiate photosynthesis [1-5]. Among them the foliose species seem to be more effective in low temperature protection [6]. The Antarctic lichen Umbilicaria aprina reveals the lowest detected photosynthetic activity [1]. The spontaneous dehydration of thallus is a way to resist very low temperature experienced by lichens, therefore both drought and cold resistance may have similar molecular mechanism. The formation of molecular glass may allow cell to survive deep dehydration [7].

The understanding of the molecular mechanism of the metabolic activity recovery during rehydration of thallus requires the knowledge on a number and distribution of water binding sites, sequence and kinetics of their saturation, and the formation of tightly and loosely bound water fractions at different steps of hydration process. The rehydration process may be accompanied by the dissolving of the water soluble solid fraction, and by the swelling of the system [8].

The thalli of fruticose lichen Cetraria aculeata (Schreb.) Fr. were collected in Arctowski Polar Station, King George Island, Maritime Antarctic. Proton FID is a superposition of the solid signal and two liquid signal components coming from tightly bound ( ≈∗

2T 100 µs) and loosely bound water fraction ( ≈∗2T 1000

µs). Gaussian function yields satisfactory approximation of solid signal for hydrated thalli at higher temperature region, however for the samples at lower hydration levels much better fits supplies Abragam function (with the line halfwidths equal to 34 kHz). Such a form of solid signal component is characteristic for carbohydrate systems forming molecular glass [9]. The liquid component of NMR signal decays smoothly in intensity with the temperature, suggesting that in the tested range of hydrations (∆m/m0 between 3.9% to 19.9%) no cooperative water freezing takes place. The amount of free water is not sufficient to initiate the ice crystallization process. Address for correspondence: H. Harańczyk, D. Sc., Institute of Physics, Jagiellonian University, ul. Reymonta 4, 30-059 Cracow, e-mail: hubert.haranczyk@uj.edu.pl References: [1] H. Harańczyk „On water in etremely dry biological systems”, Wyd. UJ 2003 pp. 276. [2] H. Harańczyk, J. Grandjean, M. Olech, Colloids & Surfaces, B: Biointerfaces 28/4, 239, (2003). [3] H. Harańczyk, J. Grandjean, M. Olech, M. Michalik, Colloids & Surfaces, B: Biointerfaces 28/4, 251, (2003). [4] H. Harańczyk, A. Pietrzyk, A. Leja, M.A. Olech, Acta Phys. Polon. 109, 411 (2006). [5] H. Harańczyk, M. Bacior, P. Jastrzębska, M.A. Olech, Acta Phys. Polon. A115, 516 (2009). [6] H. Harańczyk, M. Bacior, M.A. Olech, Antarctic Science 20, 527 (2008). [7] T. Kikawada, N. Minawaka, M. Watanabe, T. Okuda, Integr. Comp. Biol. 45, 710 (2003). [8] H. Harańczyk, J. Czak, P. Nowak, J. Nizioł, “Initial phases of dry DNA rehydration by NMR and sorption isotherm”, Acta Phys. Polon. in press (2010). [9] W. Derbyshire, M. van den Bosch, D. van Dusschoten, W. MacNaughtan, I.A. Farhat, M.A. Hemminga, J.R. Mitchell, J. Magn. Res. 168, 278 (2004).

13

MAPPING OF THE B1 FIELD USING ADIABATIC EXCITATION PULSES

Franciszek Hennel

Bruker-BioSpin MRI, Ettlingen, Germany

Introduction. The knowledge of map of the radio-frequency field (B1) is important for numerous MRI applications, such as the position-dependent flip-angle calibration, homogenizing of the B1 field with transmit arrays (“B1-shimming”) or the acceleration of multi-dimensional selective RF pulses (“Transmit-SENSE”). Existing B1-mapping methods use either the intensity or the phase of the signal as the source of information. The phase-based approach (1,2) has the advantage of being independent on the relaxation properties of the object, and, as recently showed by Morrell, achieves a better sensitivity than in signal amplitude-based methods (3). Morrell’s method uses two RF pulses: the first one producing a B1-dependent nutation about the x-axis, the second one rotating this state to the x-y plane and thus linking the nutation angle with the signal phase. The dynamic range of this method is limited by the ability of the second pulse to generate transverse magnetization: its effect is best at 90°, and vanishes at 180°. We propose a way of generating the B1-dependent signal phase with higher dynamic range using an adiabatic half passage excitation pulse. Methods. It has been demonstrated (4) that an inverse adiabatic half passage pulse (IAHP), with the sweep starting on-resonance, also allows deriving B1 maps from the phase of the signal. This required simulation-based lookup tables and was prone to errors due to resonance offsets. The improved version presented here uses two IAHP’s, each of them preceded by a short block section without the frequency sweep. The phase of this block pulse is identical with the starting phase of the IAHP in one experiment, and the opposite in the other. The phase difference of the images acquired in the two experiments is affected solely by the block section and allows a straightforward calculation of the RF field strength: B1 = ∆φ/2γτ, where τ is the block pulse duration. As a further, optional modification, each of the IAHP pulses can be preceded by a block rewinder pulse of the phase opposite to that of the IAHP. Its role is to compensate the phase accrual caused by the adiabatic pulse itself. Although this phase is subtracted upon the phase difference calculation, it may lead to intra-voxel dephasing (and signal loss) in regions of strong B1 gradients. The duration of the rewinder pulse was numerically optimized for the expected range of B1 and the given IAHP shape and sweep. As an alternative way of rephasing, an adiabatic full-passage (AFP) pulse with double duration, half amplitude and half sweep has been used to produce a spin echo. All excitation schemes have been combined with a standard 3DFT imaging gradient sequence. Bloch equations have been numerically solved for all sequences to verify the B1 vs. ∆φ dependence and its sensitivity to Bo offsets. Results. The result of the simulations (Fig. 1), for which a 5ms cos-sine IAHP pulse of 5kHz sweep was taken, show a good agreement of the phase difference with the theoretical value for an astonishingly high range of B1 values. The expected linear dependence is seen even at B1/2 = 1kHz, where the adiabadicity factor (nutation- to nutation-axis-velocity ratio) is of only 0.6. In the same range, the sensitivity to Bo offsets becomes marginal. All variants of the method were used to map the B1 field of a 3cm surface RF coil on a 7T Bruker BioSpec system. The phase difference was unwrapped and scaled to provide an image of B1 in frequency units. For a verification, another image was measured with a rectangular 1 ms pulse of the same peak

14

amplitude as the IAHP. This pulse produces black bands where B1/2 = n ×500 Hz, i.e., where the effective flip angle is a multiple of 180 degrees. An excellent agreement with measured B1 contours could be observed. (Fig. 2). A reduction of signal, and artifacts in the B1 map in a small region close to the RF coil circuit could be observed and disappeared when the rewinder pulse or the spin echo sequence was used.

Discussion/Conclusion. Compared to (2) our method replaces the second RF pulse by an adiabatic 90 degree plane rotation. This guarantees that the nutation angle caused by the initial pulse (block section) is perfectly transferred to the signal phase in a high range of B1 values. As a result, the B1 map can be reconstructed without lookup tables and in a higher dynamic range, making the method attractive for the evaluation of surface coils. The application of a rewinder RF pulse compensates the dephasing caused by the IHAP, similarly to the matched-AFP echo. The inherent 90-degree flip angle of this method has to be admitted as a drawback in the context of fast 3D acquisition. References: 1. C.H. Oh et al, Magn. Reson. Imaging, 8, 21-25 (1990) 2. G. R. Morrell, Magn. Reson. Med. 60, 889-894 (2008) 3. G. R. Morrell, ISMRM 2009, 376 4. F. Hennel et al., ISMRM 2009, 2610

Fig.1. Simulated dependence of the phase difference (vertical, rad) as a function of B1 (kHz) for a range of B0 offsets (±500Hz).

Fig.2. Measured 500 Hz contours of the B1 field cross-checked with a Nx180deg signal nulling for a 1ms block pulse.

15

THE MULTIPARAMETRIC CMR-BASED ASSESSMENT OF HEART FUNCTION IN MURINE MODEL OF HEART FAILURE TGaq*44

Magdalena Jabłońska1,2, UrszulaTyrankiewicz1, Tomasz Skórka1, Henryk Figiel2, Sylwia Heinze-Paluchowska1, Mirosław Woźniak3, and Stefan Chłopicki3

1Department of Magnetic Resonance Imaging, H. Niewodniczanski Institute of Nuclear Physics PAN, Krakow, Poland; 2Department of Medical Physics and Biophysics, AGH University of Science and Technology, Krakow, Poland; 3Department of Experimental

Pharmacology, Chair of Pharmacology CMUJ, Krakow, Poland

Introduction: The aims of this work was to assess the feasibility of the selected explorative data analysis techniques for characterization of the murine model of heart failure (TGaq*44) and to find homogeneous groups which cluster mice with regard to the mechanisms present on current stage of pathology progression. Methods: CMR-based images of the left ventricle were created in the short axis plane at the papillary muscles level and then segmented semi-automatically for evaluation left ventricle area and its plot against the time. Dobutamine induced stress was used to unmask potential alterations in cardiac function at early stage of heart failure, so the cardiac function was measured twice: at the rest and after dobutamine administration. As the indicators of heart function some parameters which describe the systolic and diastolic phase of cardiac cycle were estimated. For the data analysis, methods of unsupervised classification (cluster analysis) were used. This method allows for using descriptive modeling and exploring information about character of sample or population. Results and conclusions: In this work, abilities of multi-parametric heart function differentiation method facilitate the assessment of induced stress response. This approach seemed to be adequate with regard to the complicated mechanisms existing in this kind of the cardiac dysfunction being developed by TGaq*44 transgenic murine strain. Such dysfunctions can’t be recognize by single variable observation or comparison and need more sophisticated approaches. The investigation of changes at stress, supported by explorative techniques, should give better chance for separating homogeneous groups as a preliminary method of data analysis which provides to simplifying and ordering techniques of results interpretation and information searching. Acknowledgements: This work was supported by PMSHE (grant NN 518419733).

16

MRI APPLICATION OF MICROSTRIP RF COILS

Krzysztof Jasiński1, Peter Latta2, Vyacheslav Volotovskyy2, Anna Młynarczyk1, Władysław P.Węglarz1, and Bogusław Tomanek1,2

1Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland; 2Institute for

Biodiagnostics, National Research Council of Canada, Winnipeg, Canada

A microstrip is made of a metallic strip on PCB surface creating a planar RF transmission line, commonly used in microwave electronics. The RF coils based on microstrip design, have been already applied to MRI and MRS [1,2]. These coils produce homogenous RF field only within a very restricted field of view. We present here application of microstrip microcoil to MR microscopy.

To asses MRI performance of a microstrip, numerical simulations were performed. Finite Element Method, full 3D quasi static, time harmonic simulations showed a decrease of the RF field amplitude with the distance from the coil.

The simulation software provided impedance of the coil, spatial distribution of B1 field and current density which allowed calculation of intrinsic SNR for many configurations of microcoils, enabling selection of an optimal microstrip coil geometry.

To test the coil performance microstrip coil was constructed, tuned to 500 MHz and matched to 50 Ohm. Electric parameters measured on-the-bench agreed with numerical simulations.

For MR imaging experiments, the microstrip coil was positioned inside the 11.7T vertical bore magnet (Oxford Instr. UK) equipped with a commercial 72mm ID gradient set (maximum 600 mT/m) and Avance console (Bruker, Germany).

Several MR images were acquired proving the high performance of the microstrip microcoil for MRI microscopy.

References: [1] X. Zhang, K. Ugurbil, W. Chen, J. Magn. Reson. 161 (2003) 242–251. [2] P.J.M. van Bentum, J.W.G. Janssen, A.P.M. Kentgens, J. Bart, J.G.E. Gardeniers, J. Magn. Reson. 189 (2007) 104–113.

17

TAUTOMERISM OF SCHIFF BASES DERIVED FROM (S)-DIPHENYLVALINOL: THE SOLID STATE NMR

AND X-RAY STRUCTURE STUDIES

Magdalena Jaworska1, Paweł B. Hrynczyszyn1, Katarzyna Nowicka2, Włodzimierz Ciesielski2, and Marek J. Potrzebowski2

1 Wydział Chemii, Uniwersytet Mikołaja Kopernika, Toruń, Poland;

2 Centrum Badań Molekularnych i Makromolekularnych, Polska Akademia Nauk, Łódź, Poland

Schiff bases can be obtained in condensation of aromatic or aliphatic primary amines

with aldehydes or ketones. The tautomerism of o-hydroxy Schiff bases is a very important phenomena: 1) in biological processes which are concerned in less stable tautomers responsible for biochemical acivity [1], 2) in catalytic systems often used in asymmetric C-C bond formation reactions including addition of diorganozincs, cyanosilylation of carbonyl compounds, nitroaldol reaction, alkynylation etc. Theoretically tridentate o-hydroxy Schiff bases can exist in two tautomeric forms: phenol-imine and keto-amine. (S)-Diphenylvalinol (1) was obtained from L-valine according to method previously reported by Itsuno [3]. A series of eight tridentate Schiff bases was prepared in reaction of (1) with different salicylaldehydes and 2-hydroxy-1-naphtaldehyde in ethanol at room temperature. All compounds were yellow or orange crystalline solids.

Scheme 1: Synthesis of Schiff bases from (S)-diphenylvalinol

The crystals of 2a and 2e suitable for the diffraction experiment have been obtained from EtOH/water solution and X-ray structures were prepared. The significant differences of some bond lengths have been noticed which was concerned in tautomerism of 2e assisted by intramolecular hydrogen bond between two hydroxyl groups. This phenomena was confirmed by CP MAS NMR (13C and 15N) and PASS-2D experiment. Experimental data were compared with simulation on WinMas (based on Herzfeld-Berger algorithm). The principal values of δ11, δ22 and δ33 of the chemical shift tensor were calculated as well as isotropic average (δiso), Ω and κ for carbons and nitrogen atoms which confirmed the occurrence of tautomeric keto-amine form for compound 2e.

References: [1] E. D. Raczyńska, W. Kosińska, B. Ośmiałowski, R. Gawinecki, Chem. Rev. 2005, 105, 3561-3612. [2] for example.: Y. L. Bennani, S. Hanessian, Chem. Rev. 1997, 97, 3161-3195. [3] S. Itsuno, K. Ito, A. Hirao, S. Nakahama, J. Org. Chem., 1984, 49, 555-557.

EtOH

Ph

Ph

OH

N

H

OH

R1

R2

R3

H2N

Ph

OH

Ph

2, gdzie:

a: R1=R2=R3= H b: R1= t-Bu, R2= H, R3= t-Bu

c: R1= H, R2= N,N-dietyloamino, R3= H d: R1= H, R2= H, R3= Br

e: R1= OH, R2= H, R3= H f: R1= Me, R2= H, R3= H

g: R1= i-Pr, R2= H, R3= H h: R1= H, R2 and R3 = phenyl

salicylaldehydes or 2-hydroxy-1-naphtaldehyde

(S)-diphenylvalinol

18

ADDUCTS FORMATION OF RHODIUM(II) TETRACARBOXYLATES WITH METHIONINE DERIVATIVES: INVESTIGATIONS

BY THE USE OF VIS AND NMR TECHNIQUES

Jarosław Jaźwiński and Rafał Głaszczka

Institute of Organic Chemistry, Polish Academy of Sciences Warszawa, Poland

Dinuclear rhodium(II) salts have unique paddlewheel structure with Rh-Rh single bond

surrounded by four carboxylates1. Dirhodium salts are soft bases and can accept one or two organic ligands with oxygen, sulphur, phosphorus or nitrogen atoms in axial positions forming 1:1 and 1:2 adducts. Complexes of rhodium(II) salts with organic ligands, obtained in situ in solution have been widely applied in chemistry, spectroscopy and medicine.2-5 Our present work concerned the investigations of rhodium(II) adducts with some derivatives of R- and S-methioninine in D2O and CDCl3 solutions, by the use of 1H, 13C, 15N NMR and VIS methods. There were three purposes of the work: (i) whether the complexation occurs in water solution, i.e in the solvent of biological importance, (ii) what is the complexation site in the ligand; (iii) what is the adduct stoichiometry.

Fig.1. Dirhodium(II) tetracarboxylates and ligands studied.

Our investigations led to the following conclusions: methionine, methionine hydrochloride and methionine methyl ester hydrochloride formed the adducts with rhodium(II) tetraacetate in water solution; VIS titration experiments revealed stepwise formation of two adducts, 1:1 and 1:2. Examination of adduct formation shifts (1H and 13C NMR) showed that dirhodium teracarboxylates bonded ligands via sulfur atoms. Amide and imide derivatives of methionine, insoluble in water, behaved similarly in CDCl3 solution. References:

1. Cotton F.A., Hillard E.A., Murillo C.A., J. Am. Chem. Soc. 2002, 124, 5658; 2. Agaskar A., Cotton F.A., Falvello L.R., Han S. J., Am. Chem. Soc. 1986, 108, 1214; 3. Díaz Gómez E., Duddeck H., Magn. Reson. Chem. 2007, 46, 23; 4. Díaz Gómez E., Albert A., Duddeck H., Kozhushkov S. I., Meijere A., Eur. J. Org. Chem. 2006,

2278; 5. Jaźwiński J., J. Mol. Struct., 2005, 750, 7.

R1 = CH3, CF3, C(CF3)(OCH3)(C6H5)

O O

R1

O O

R1

Rh

OO

R1

Rh

O O

R1

O

N

SO

R2

R3

R4

R5

ep* – electron pair

Ft** =

O

O

R2

R3

R4

R5

H H H ep*

CH3 H H ep*

CH3 H CHO ep*

CH3 CH3 CH3 ep*

H R3 = R

4 = Ft** ep*

H R3 = R

4 = Ft** O

19

MOLECULAR DYNAMICS AND STRUCTURE IN DIBLOCK COPOLYMERS STUDIED BY NMR AND BROADBAND DIELECTRIC

SPECTROSCOPY

Jacek Jenczyk, Monika Makrocka-Rydzyk, Aleksandra Wypych, Stanisław Głowinkowski, and Stefan Jurga

Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University,

Poznań, Poland

Block copolymers are under great research interest due to their current and potential applications. Properties of these systems depend on individual blocks characteristics and on the interaction between the components[1]. We have investigated three nearly symmetric poly(styrene-b-isporene) diblock copolymers: PS(11500)-b-PI(10500)→SI1, PS(45000)-b-PI(46000)→SI2 and PS(135000)-b-PI(131000)→SI3. Nuclear Magnetic Resonance and Broadband Dielectric Spectroscopy studies were applied to characterize molecular motions in copolymers and their neat components. It appeared that the flexible polyisoprene (PI) and stiff polystyrene (PS) blocks in copolymer influence one another in terms of molecular dynamics. As a result the increase of the glass transition temperatures (Tg) for PI chains for all copolymers was observed [2]. Moreover, there is a substantial broadening in the distribution of relaxation times connected with the normal mode and glass transition process in copolymers in comparison with neat PI and it is more pronounced for the copolymer of higher molecular weight. It is assumed that these changes result from both type chains mutual interactions, since PS blocks make spatial confinement for PI chains motion, whereas the moving PI chains act as plasticizer for PS blocks. The size of PS domains were determined by NMR spin diffusion experiment. The obtained size domain values are in good agreement with those determined by other experimental techniques. [3,4]. References: [1] I.W. Hamley Developments in Block Copolymer Science and Technology (2004) John

Wiley&Sons, Ltd. [2] J. Jenczyk, M. Makrocka-Rydzyk, A. Wypych, S. Głowinkowski, M. Radosz, S. Jurga J.

Non Cryst. Sol. (2009), accepted for publication. [3] B. Cott Pinheiro, K. I. Winey Macromolecules 31 (1998) 4447-4456 [4] J. Denault, B. Morese-Seguela, J. Prud’homme Macromolecules 23 (1990) 4658-4670 Acknowledgements: This work is supported by 6th Framework Programme under SoftComp Grant No 502235-2 and research grant No N N202 128536 (Poland). A. Wypych expresses her appreciation to the Ministry of Science and Higher Education in Poland for a Postdoctoral Fellowship (POL-POSTDOC III).

20

BACKBONE DYNAMICS OF TFE-INDUCED NATIVE-LIKE FOLD OF REGION 4 OF INITIALLY UNFOLDED E. coli RNA POLYMERASE

σ70 SUBUNIT

Piotr Kaczka1, Agnieszka Polkowska-Nowakowska1, Krystyna Bolewska1, Igor Zhukov1,2, Jarosław Poznański1, and Kazimierz L. Wierzchowski1

1Institute of Biochemistry and Biophysics PAS; Warszawa, Poland; 2Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia

Folding of a recombinant protein rECσ70

4, containing domain 4 of E. coli RNA polymerase σ70 subunit, accompanying addition of 2,2,2-trifluoroethanol (TFE) to its aqueous solution, was monitored by heteronuclear NMR spectroscopy. TFE-induced migration of the resonance signals in a series of 15N HSQC spectra displayed sequence-dependent heterogeneity. A common trend of uniform upfield shift in both 1H and 15N dimensions, indicative of generation of helical structures, breaks down for some residues at 10-15% TFE (v/v) strongly suggesting the buildup of non-helical regions separating initially induced helices. Spontaneous organization of the helical regions of the polypeptide into a 3D-structure, revealed from structural constraints deduced from 15N- and 13C-edited NOESY spectra, accompany secondary structure formation. The absence of long-range cross-peaks in 15N-edited NOESY spectra recorded for low pH and at 10% (v/v) TFE protein solutions clearly indicates that in these conditions the live-time of the low-populated high-order structures is relatively short. Contrary for 30% TFE protein solution 23 structural cross-peaks in 15N- and 13C-edited NOESY spectra were unequivocally assigned, strongly suggesting that high TFE solution stabilizes protein tertiary structure. Analysis of CSI descriptors estimated for NMR spectra recorded at 10% and 30% TFE allowed estimation of the population of the folded rECσ70

4. The sequential distribution of TFE-induced secondary chemical shifts clearly indicates that 2,2,2-trifluoroethanol acts not only as a canonical helix inducer, but also tends to stabilize a spatial arrangement of the induced secondary structure elements. The postulated HLHTH folding pattern perfectly agrees with the sequential distribution secondasry structure elements.

Analysis of 15N relaxation parameters pointed some regions exhibiting significant deterioration in fast internal motion in favor of the slower ones. The whole HLHTF fragment, embracing the H1, H2, and H3 helices and the intervening loop/turn sequences, displays the increased values of the spectral density function J(0) accompanying the decreased values of spectral density at high frequence J(0.87ωH), both indicative for a reduced backbone flexibility on the pico- to nanosecond time scale and large contributions from slow motional process on the micro- to millisecond time scale. This supports the hypothesis that the helical secondary structures become formed cooperatively and exhibit high propensity to fold into a relatively stable tertiary structure. Summarizing, analysis of the relaxation parameters demonstrated that at 30% TFE solution the motion of the rECσ70

4 backbone, with the exception of the terminal residues, is considerably restricted, as indicated by the values of the generalized order parameter, showing strong tendency of rECσ70

4 to fold. Reasonable and relatively not uniform values of S2 suggest that rECσ70

4 exhibit anisotropic behavior. All together, indicate that TFE induces not only the secondary helical structures, but also a tertiary one resembling in organization that found in homologous σ4 domains.

21

fMRI QUALITY ASSURANCE SYSTEM: PHANTOM AND AUTOMATIC DATA ANALYSIS

Karolina Kamińska1, Stanisław Adaszewski1, Ewa Piątkowska-Janko1,

Piotr Bogorodzki1, and Maciej Pisklak2

1Institute of Radioelectronics,Warsaw University of Technology, 2Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland

Introduction: In typical functional MRI (fMRI) experiments on 1,5T scanners, blood oxygenation level dependent (BOLD) signal changes are in the range of a few percent [1]. Hence, system stability during fMRI acquisition is essential for repeatedly and reliable results. The main aim of this work was to create a fMRI quality assurance (QA) system for daily scanner performance monitoring. Proposed system consists of: a) agar head phantom mimicking MRI RF coil loading caused be human head, b) scanning protocol with typical for fMRI studies settings and automatic image analysis software. Subjects and Methods: The head phantom for QA analysis is a 17cm sphere filled with a doped agar. The purpose of a doping was to mimic healthy brain relaxation times. We adopted original (Functional Imaging Research Schizophrenia Testbed - Biomedical Informatics Research Network) FIRST-BIRN agar phantom recipe [2] in order to reach target relaxation times T1 = 600 ms, T2 = 100 ms (for concentration 0,96% of agar and 1,77mM NiCl2 [3]). The phantom relaxation times were evaluated on 1,5 T scanner with the following settings: Multi-Echo TE= 30, 60, 90, 120 ms, TR = 1 s and Inversion Recovery TI = 100, 200, 400, 800, 1600, 2400 ms, TR = 10 s. T1 and T2 maps were generated using home written Matlab script. An automatic analysis software called QAWizzard was developed in C++ using open-source build system Cmake and libraries, GDCM – a cross-platform library for DICOM medical files, SQLite - SQL database engine, wxWidgets - a widget toolkit for creating graphical user interfaces, VTK – an open source graphics toolkit. QAWizzard describes the stability of the MR scanner giving parameters: a) drift, which defines the signal changes during acquisition, b) signal to noise ratio (SNR) and signal to fluctuation ratio (SFNR) [2]. Results: Relaxation times of proposed phantom were evaluated on a 1,5 T Siemens scanner with setting as described earlier. A relaxation maps show good homogeneity either T1 or T2 parameters. A mean relaxation times was calculated for centrally located 10 cm3 volume giving T1 = 560 ms and T2 = 90 ms. Proposed system was evaluated on three 1,5T MRI scanners. The results of 14-days long trials are summarized in Tb1. Medical Center

Mean signal value [a.u.] SNR [a.u.] SFNR [a.u.] Drift [a.u.]

1 1238,5± 23,89 130,62± 8,68 129,03± 3,09 -0,00067± 0,00064 2 1198,49± 20,74 216,21± 8,04 214,54± 4,21 -0,00034± 0,00083 3 6553,4± 580,39 311,77± 21,46 233,07± 5,127 -0,03333± 0,00193

Table 1. QA data

22

Discussion/Conclusion: Scanner stability is a key to successful fMRI research. Our example shows that QA system is a useful tool for scanner stability monitoring. Regular measurements of SNR, SFNR and signal drift provide important feedback about scanner performance. References: [1]Matthews, P.M., 2001, Oxford University Press,p 3-34; [2] Friedman L., Glover G.H., 2006, J. Magn. Reson. Imaging, 23, p.827-839; [3] Tofts, P.S., 2003, John Wiley & Sons, Ltd, p. 55-81.

23

NMR RELAXATION MEASUREMENTS OF OXIDATION PROCESSES IN BLOOD SERUM

Joanna Kamińska, Jan Kobierski, and Barbara Blicharska

Jagiellonian University, Department of Physics, Krakow, Poland

Oxidation reactions are crucial for life. One of reactive oxygen species: hydrogen peroxide can be produced and consumed in cells. At low concentration aqueous solution of H2O2 is widely used for bleaching and disinfection (Fenton reaction). NMR relaxation investigations of Fenton reaction in blood serum may help to understood mechanism of oxidation of biological systems.

In this communication we present the results of time-changes of relaxation times in blood serum after addition of H2O2. It is interesting that behavior of spin-lattice T1 and spin-spin T2 relaxation times is different when in blood serum additionally the antioxidants like Vit.C and Vit.E are present.

24

THE USEFULNESS OF DIFFUSION-WEIGHTED IMAGING IN DISTINGUISHING BETWEEN SOME BRAIN PATHOLOGIES

Aleksandra Klimas1, Zofia Drzazga1, and Ewa Kluczewska2

1Department of Medical Physics – University of Silesia, Katowice, Poland; 2Department of Clinical Radiology and Diagnostics Silesian University of Medicine, Katowice, Zabrze, Poland

Introduction Diffusion-weighted imaging (DWI) has become a powerful, multifaceted tool for advanced imaging in diagnosis and treatment planning. Diffusion MRI measures the mobility of the water protons and thus provides a window into tissue microstructure. DWI is used in imaging the central nervous system (CNS) and quantitatively characterizing different pathological states of brain (1,2,3) The aim of this work is to test the ADC values as a function of b-value in distinguishing between some brain pathologies, such as: schwannoma acoustic neuroma, apoplectic lesion, sclerosis multiplex, arachnoid cyst.

Materials and methods T1, T2-weighted spin echo sequence and DWI of 4 patients (2 women, 2 man) with different brain diseases and two healthy volunteers were performed on 1,5 T GE MR unit. DWI was performed using spin echo EPI with diffusion gradient applied in three orthogonal directions with b=500 s/mm2, b=1000 s/mm2, b=1200 s/mm2 and additionally with b=0 s/mm2. The ADC maps were calculated using Functool on the GE console. Results and Discussion Figure 1 presents ADC map with regions-of-interest (ROIs) marked manually on slices of patients with: a) schwannoma acoustic neuroma, b) apoplectic lesion, c) sclerosis multiplex, d) arachnoid cyst. We can see that regions of diseases are characterized by increase of signal intensity dependent on pathology.

A) C)

B) D)

Figure 1. ADC map with regions-of-interest (ROIs) a) schwannoma acoustic neuroma, b) apoplectic lesion, c) sclerosis multiplex, d) arachnoid cyst.

25

Figure 2. The ADC values for different pathologies and normal tissue. Insert: Comparison of ADC values of normal brain tissue between patients and healthy volunteers. The calculated ADC values as a function of b-value in ROIs are presented in Figure 2. One can see that ADC values are only slightly dependent on b-values. For healthy brain tissue ADC parameter is below 1.0 x 10-^3 mm2/s similarly as in other works (4). The highest values of ADC (about 2,5^x10^-2 mm2/s) was found for arachnoid cyst and apoplectic lesion what is connected with strong hydration of studied tissue. Smaller changes of ADC parameters are observed for schwannoma acoustic neuroma and sclerosis multiplex. Insert in Figure 2 shows comparison between ADC values calculated in areas outside the pathology of patients and in ROIs of healthy volunteers. It should be noted that calculated values of ADC are similar in the range of error. It means that it is possible to analyze pathologic changes respect to suitable healthy part of the brain.

∆ADC = ADChealthy – ADCpathology b-value sclerosis multiplex schwannoma acoustic neuroma apoplectic lesion arachnoid cyst

500 45,6 86,2 160,7 163,0 100 50,0 90,1 163,7 172,7

1200 44,2 90,0 161,0 175,7

Table 1. Differences between ADC values in the normal and pathological tissue of the brain. In Table 1 differences between ADC values in the normal and pathological tissue of the brain (∆ADC = ADChealthy – ADCpathology) are listed. Quantitative results confirm that the highest change of the signal intensity was found for arachnoid cyst and the smallest for sclerosis multiplex and this parameter is nearly independent on b-value. Conclusions: ∆ADC values obtained from ADC maps for lesion and normal areas of the brain could be useful for distinguishing between different pathological states.

References: 1. Lutsep HL, Albers GW, de Crespigny A, Kamat GN, Marks MP, Moseley ME. Ann Neurol 1997;41:574–580. 2. Werring DJ, Clark CA, Barker GJ, Thompson AJ, Miller DH. Neurology 1999;52:1626–1632. 3. Mauricio Castillo, J. Keith Smith, Lester Kwock, and Kathy Wilber.AJNR Am J Neuroradiol 2001; 22:60–64. 4. L. Røhl1, J. Sørensen2, A. Obel1, L. Elgaard1, L. Bro1, E. Nielsen1, A. Nehen1. Proc. Intl. Soc. Mag. Reson. Med. 11 (2004).

26

CHEMICAL EXCHANGE PROCESSES IN HYDROGEN PEROXIDE SOLUTIONS

OBSERVED BY NMR RELAXATION

Jan Kobierski1, Hartwig Peemoeller2, and Barbara Blicharska1

1Jagiellonian University, Department of Physics, Krakow, Poland; 2University of Waterloo, Department of Physics, Waterloo, ON, Canada

It is known, that hydrogen in aqueous solution of H2O2 exchanges quickly and completely between hydrogen peroxide and water. This process is called a chemical exchange. NMR experiments in high fields enable to observe a chemical exchange. We have used Selective Inversion Recovery method and 2D experiments to estimate the rate of this process.

In some cases T1ρ dispersion measurements can be applied not only for calculation of molecular dynamics parameters (like correlation time and activation energy but also for estimation of time of exchange processes. If exchange time is shorter than correlation time the relaxation is determined by the time of chemical exchange. In this communication we present application of NMR methods for observation of chemical exchange for H2O2 solution samples.

27

DETECTOR FOR PROTON-ELECTRON DOUBLE RESONANCE IMAGING (PEDRI)

Łukasz Kołaszewski1, Piotr Bogorodzki1, Ewa Piatkowska-Janko1, Jerzy Piotrowski2,

Jerzy Skulski2, and Maciej Pisklak3

1Institute of Radioelectronics, Faculty of Electronics and Information Technology at the Warsaw University of Technology, POLAND; 2Institute of Microelectronics and Optoelectonics, Faculty of Electronics and Information Technology at the Warsaw University of Technology, POLAND; 3Department of Physical Chemistry, Faculty of Pharmacy at the Medical University of Warsaw,

POLAND Purpose/Introduction PEDRI makes use of a double resonance technique based on the Overhauser-Effect [1], which is essentially a combination of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Simultaneously irradiation at EPR frequency during NMR signal acquisition can cause a transfer of magnetic polarization from the electrons to the nuclear spins, resulting in an enhancement of the measured NMR signal up to 330 times [1]. Subjects and Methods Detector was designed to Bruker BNT1000 magnetic field of 89 mT. This results in proton Larmor's frequency 3.728 MHz and 2.454 GHz for electrons. Due to very high electron's gyromagnetic ratio 28 GHz/T [2], compared to proton's 42.6 MHz/T [2], the microwaves rectangular resonator has been used. The resonator works in mode TE102, providing mutual perpendicularity of three magnetic fields ( BEPR, BNMR, B0 ). In a geometrical center of the resonator a NMR probe (21 turns of 0.9 mm2 copper wire solenoid coil) is placed. The probe is connected via impedance transformer to scanner T/R switch. A 1.4 ml teflon container filled with 4.0 mM water solution of TEMPOL free radical was placed inside the probe. Thanks to TE102 mode, there is minimal E field component within sample volume, which prevents it's heating. Results A microwave resonator was measured on AgilentTechnology E5071C vector network analyzer giving following results: unloaded f0=2.45563 GHz, Q=4464 and loaded with sample f0=2.45417 GHz, Q=2231. A NMR probe was tuned to 3.7284 MHz, with measured Q-factor about 90. Thereafter a whole detector was placed in Bruker BNT1000 resistive magnet bore, with mean magnetic induction of 89 mT and linear sweep of +/-0.82 mT. Signal generator HP33120A was used to control field sweep and network analyzer via optical isolation. Network analyzer was used as a receiver in the time domain mode, measuring resonator's S11 factor during field sweep. Total spectrum acquisition time was 12 minutes with total 36 averages of 20 seconds lasting acquisitions. Obtained spectrum is similar to the one obtained on MiniScopeMS200 spectrometer with there are three absorption lines, which are characteristic for TEMPOL radical [2]. However half height width 1.63 MHz was substantially smaller. Discussion/Conclusion Proposed detector was tested on BrukerBNT1000 with good results. Both NMR and EPR blocks are working as they was indented and it makes potential ability for PEDRI idea. References: [1] "Design and evaluation of Overhauser enhanced MRI visible markers"; Raimo Joensuu; [2] "Dynamic Nuclear Polarization with Nitoxides Dissolved in Biological Fluid"; Daniel Grucker;

28

RECENT ADVANCES IN MULTIDIMENSIONAL NMR SPECTROSCOPY WITH SPARSE RANDOM SAMPLING

Wiktor Koźmiński, Krzysztof Kazimierczuk, Maria Misiak, Jan Stanek,

and Anna Zawadzka-Kazimierczuk

Faculty of Chemistry, University of Warsaw, Warszawa, Poland

Nuclear Magnetic Resonance is nowadays one of the most efficient spectroscopic techniques, providing insight into molecular structure and dynamics. The observed frequencies of oscillatory Free Induction Decay signal are sensitive indicators of electron surroundings of nuclei and their, often very subtle, changes caused by inter- and intramolecular interactions. However, especially in studies of biomolecules, complexity of NMR spectra often causes difficulties in their interpretation. This problem may be solved by acquisition of multidimensional spectra which increases resolution and enables identification of nuclei connected by mutual interactions.

The conventional approach to recording multidimensional NMR experiments is limited by the need for fulfilling of the Nyquist Theorem to avoid aliasing. It determines the sampling rate to be twice higher than the highest frequency expected in the signal. In consequence, this is an implicit limit for the maximum evolution time and therefore obtainable resolution. Because of this, for spectra of higher dimensionality, it is practically impossible to reach the relaxation limits in indirectly sampled dimensions. The problem of effective acquisition of multidimensional NMR spectra is getting relatively more important at high fields, where the required Nyquist rate increases together with spectral width.

Random sampling of evolution time space and Multidimensional Fourier Transform, enables to obtain, without aliasing, spectra at a very small fraction of number of data points required conventionally. Moreover, the relative level of artifacts caused by random sampling does not depend on this fraction. Some new applications of high dimensionality (3-6D) will be shown where the maximum evolution times are limited only by transverse relaxation rates. References: K. Kazimierczuk, W. Koźmiński, I. Zhukov, J. Magn. Reson. 179, 323-328 (2006). K. Kazimierczuk, A. Zawadzka, W. Koźmiński, I. Zhukov, J. Biomol. NMR, 36, 157-168 (2006) M. Misiak, W. Koźmiński, Magn. Res Chem., 45, 171-174 (2007) K. Kazimierczuk, A. Zawadzka, W. Koźmiński, I. Zhukov, J. Magn. Reson., 188, 344-356 (2007) K. Kazimierczuk, A. Zawadzka, W. Koźmiński, J. Magn. Reson. 192, 123-130 (2008) K. Kazimierczuk, A. Zawadzka, W. Koźmiński, I. Zhukov, J. Am. Chem. Soc., 130, 5404-5405 (2008) M. Misiak, W. Koźmiński, Magn. Res. Chem., 47, 205-209 (2009) K. Kazimierczuk, A. Zawadzka, W. Koźmiński, J. Magn. Reson., 197, 219-228 (2009)

29

THE ROLE of FULLEREN C60, CARBON NANOTUBES AND CRBON ENCAPSULATED MAGNETIC NANOPARTICLES

IN CREATING OF CREATOL (5-HYDROXYCREATININE) AND UREMIC TOXIN -METHYLGUANIDINE.

Hanna Krawczyk and Magdalena Popławska

Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland

Diabetic patients with chronic renal failure accumulate in their sera the creatinine

oxidative metabolites-the creatol (5-hydroxycreatinine), which decomposes into uremic toxin methylguanidine. The creatol was identified in the urine of patients suffering from kidney diseases in the beginning of 1990’s [1]. Its significance lies in the fact that in vivo, it is a direct product of reaction of creatinine with the hydroxyl radical [2]. Hydroxyl radical has a highest reactivity among various species of free radicals. It is known that in position 5 of creatinine in oxidative conditions, one of the protons at C-5 of the imidazole ring is substituted with a group containing an oxygen atom [3-5] or amine group [3]. Such transformation proceeds with the presence of activated carbon[5]. This observation is important as the activated carbon coated with different types of polymer is widely used as a sorbent in haemoperfusion techniques for the removal of various toxic products and metabolites [6]. Taking this data into account, we investigated products of creatinine reaction with water in the presence of other forms of carbon- fulleren C60, carbon nanotubes (CNTs) and carbon encapsulated magnetic nanoparticels (CEMNPs).

Reference:

1. K. Nakamura and K. Ienaga Experientia 1990, 46, 470-472. 2. K.Nakamura, K.Ienaga ,T. Yokozawa, N. Fujitsuka Nephron. 1994, 68(2):280-1 and Comment on: Nephron.

1991;58(1):42-6. 3. H. Krawczyk, A. Pietras and A. Kraska, Spectrochim. Acta A, 66(1) (2007) 9. 4. K. Nakamura; K. Kawano, (Nippon Zoki Pharmaceutical Co., Ltd., Japan). Eur. Pat. Appl. EP 1195373 A2 10 Apr 2002, 9

pp. DESIGNATED STATES: R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT, IE, SI, LT, LV, FI, RO. (English). (European Patent Organization). CODEN: EPXXDW. CLASS: ICM: C07D233-88. ICS: G01N030-02. APPLICATION: EP 2001-123251 2 Oct 2001. PRIORITY: JP 2000-303172 3 Oct 2000. DOCUMENT TYPE: Patent CA Section: 9 (Biochemical Methods) Section cross-reference(s): 14.

5. H. Krawczyk, J. Pharm. Biomed. Anal., 49 (2009) 945. 6. J.H. Henderson, C.A.McKenzie, P.J. Hilton, R.M. Leach, Thorax 56 (2001) 242–243.

30

DO ESR AND NMR DESCRIBE THE SAME DYNAMICS OF PARAMAGNETIC SYSTEMS?

Danuta Kruk

Institute of Physics, Jagiellonian University, Krakow, Poland In this contribution an attempt is made to describe within one set of parameters multi-

frequency ESR spectra for selected Gd(III) complexes and Nuclear Spin Relaxation

Dispersion Profiles (NMRD) for their solutions. So far literature reports say that such a

unified description is very difficult; in fact all attempts turned out to be unsuccessful. The

difficulties are attributed to the fact that electron and nuclear spins are sensitive to frequencies

different by three orders of magnitude and therefore a very accurate modeling of electron spin

spectral densities is needed to describe properly their wings to which nuclear spin is sensitive.

In this work instead of modeling the electron spin spectral densities we go in the direction of

an elaborated description of the relaxation processes beyond validity regimes of perturbation

approaches.

A general treatment of Electron Spin Resonance spectra and nuclear spin relaxation in

paramagnetic systems with S ≥ 1, valid for arbitrary motional conditions and interaction

strength has been applied.. This theory is known in the literature as the ‘slow motion theory’

or the ‘general theory’[1-4]. The name ‘slow motion’ indicates that the motion modulating the

relevant interaction is too slow, compared to the timescale of the spin dynamics, for the

perturbation theory to be valid. This approach is based on a full solution of the Liouville von-

Neumann equation by the multipole representation of tensor operators and the Wigner-Eckart

theorem, and has been developed and modified depending on applied motional models.

As a price for this high computational effort we succeeded for the first time with a

unified description of ESR and NMR features of paramagnetic systems.

References: 1 Westlund P.-O. Dynamics of solutions and fluid mixtures by NMR, J. J. Delpuech (Ed.), Wiley,

Chichester 1995, 173-229 2 Kowalewski J., Kruk D., Parigi G. Advances in Inorganic Chemistry, 2005, 57, 41-104 3 Kruk D., Kowalewski J. J. Chem. Phys. 2002, 116, 4079-4086 4 Kruk D., Kowalewski J., Westlund P.-O. J. Chem. Phys. 2004, 121 (5), 2215-2227 This work has been financed by Polish Ministry of Science and Education, grant No N N202 105936

31

ANISOTROPIC DIFFUSION PHANTOM FOR B-MATRIX CALCULATION

Artur Tadeusz KrzyŜak

Department of Magnetic Resonance, H. Niewodniczański Institute of Nuclear Physics PAN

Introduction Usually the components of the b matrix needed to calculate diffusion tensor (DT) are determined analytically [1]. The results are estimated only due to complex formula used in the calculation and unknown the real shapes of MRI’s gradients. We didn’t monitor e.g. the influence of addition to “cross terms” involved by eddy currents, LC circuits and other sources of gradient’s distortion. The proposed method eliminates these shortages and enables the precise and spatial determination of b matrix for any DTI sequence. Subject and method The anisotropic diffusion phantom was built in the form of a brick composed as an array of thin glass plates (100 um) separated with H2O layers (20 um). DTI measurements were performed on 1.5 T GE Signa scanner. The b matrices were calculated for selected ROIs (2mm2x1mm) of phantom’s volume for several directions of diffusion gradient vector. The calculation carried out using well-known Stejskal-Tanner equation [2],

∑∑ ==−= 3

1

3

1)

)0(

)(ln(

j ijijiDb

A

bA

where: A(b), A(0) - the echo intensities with and without the diffusion gradients, bij-components of the b-matrix, Dij - components of the known (as was assumed) diffusion tensor of phantom. In order to determine the values of b-matrix for any direction of the diffusion gradient vector, a system of no less six equations was solved for distinct Dij values. The tensor diffusion Dij values were obtained by rotation of phantom (assumption equipoise with rotation of tensor D) with various set of Euler’s angels. Data for different ROIs were compared using two tailed unpaired student’s t-test. Results The following diffusion tensor values for phantom in main axes frame D1= 1,1 +/- 0,12 [x10-3mm2/s], D2=2,22 +/- 0,21 [x10-3mm2/s], D3=2,12 +/- 0,19 [x10-3mm2/s] were registered. Preliminary example results of comparison of b matrices achieved for given ROIs for several directions of diffusion gradient vector. The statistically significant alterations (p <0.05) affirmed for some of b-matrix components for different ROIs. Conclusions The presented way [3] allows achieving all values of b-matrix for any direction of diffusion gradients and for any DTI sequences. The b matrices also can be calculated for any ROI or even every voxel of interesting space using demonstrated technique. The registered statistically significant changes for some ROIs could be caused by inhomogeneity of gradients induced by eddy currents, LC circuits, gradients coils or low precision of our measurements (e.g. manually rotations of phantom ).

References: 1. Le Bihan D. Diffusion and Perfusion Magnetic Resonance Imaging, Raven Press,1995. 2. Stejskal E.O., Tanner J.E. J. Chem. Phys., 42, 288, 1965. 3. KrzyŜak AT, UP RP, P.385276, 2008 and PCT/PL2009/000051, 2009.

32

ESR LINESHAPE FOR MULTISPIN SYSTEMS IN THE PRESENCE OF

RELAXATION Aleksandra Kubica1, Artur Mielczarek1, 2, and Danuta Kruk1

1 Institute of Physics, Jagiellonian University, Krakow, Poland 2 Instytut Fizyki Jądrowej PAN, Kraków, Poland The main theoretical difficulties in Electron Spin Resonance (ESR) spectroscopy are

caused by the fact that the electron spin system is beyond the validity range of perturbative treatments. This happens when the electron spin motions are driven by two large, non-commuting Hamiltonians; for example the Zeeman Hamiltonian, which arises from the interaction of an electron spin with a laboratory magnetic field, and the zero-field splitting interaction, which results from spin-orbit couplings for the electron spin quantum numbers S ≥ 1. We present a general treatment of ESR spectra, valid beyond the applicability of perturbative approaches, for arbitrary motions and interaction strengths. This treatment is known as the “slow motion theory” or the “general theory” [1-3]. The approach is based on expressing all relevant interactions and dynamic processes [4,5] (represented first in terms of Liouville operators) as a (super)matrix in a complete orthonormal basis set [1-3,6], including all relevant interactions and degrees of freedom of the system. The computationally heavy step is finding a small number of elements of the inverse of this complex matrix. ESR spectra are fully determined by only one element of the inverted supermatrix. The method has been extended to include other interactions, like higher terms of the zero field splitting and the anisotropy of the g-tensor. This work focuses on the complex influence of the g-tensor anisotropy on the ESR lineshapes for high electron spin quantum numbers. References: [1] Westlund P.-O. Dynamics of solutions and fluid mixtures by NMR, J. J. Delpuech (Ed.), Wiley, Chichester, 173-229, (1995) [2] Kowalewski J., Kruk D. and Parigi G. Advances in Inorganic Chemistry, 57, 41- 104, (2005) [3] Kruk D. Theory of Evolution and Relaxation of Multi-spin Systems. Application to Nuclear Magnetic Resonance (NMR) and Electron Spin Resonance (ESR), Abramis Academic, Arima Publishing UK, (2007) [4] Freed J.H., Bruno G.V. and Polnaszek C. J. Phys. Chem. 55, 5270-5281, (1971) [5] Freed J.H., Bruno G.V. and Polnaszek C. J. Phys. Chem. 75, 3385-3399, (1971) [6] Lynden-Bell R.M. Mol. Phys. 22, 837-851, (1971) This work has been financed by Polish Ministry of Science and Education, grant No N N202 105936

33

ESTIMATION OF PHOSPHOLIPIDS CONCENTRATION IN PLASMA, MONONUCLEAR CELLS, AND ERYTHROCYTES FROM PATIENTS WITH HEMATOLOGICAL CANCERS - 31P MRS IN VITRO STUDY

Małgorzata Kuliszkiewicz-Janus

Department of Hematology and Transplantology,

Wrocław Medical University, Poland

The investigations of 31P spectra of sera and cells of patients with hematological malignancies (acute leukemia, malignant lymphoma, multiple myeloma) and other cancers: renal, thyroid, esophageal are a clinical trials over the introduction of MRS to monitoring of the therapy. The changes of concentrations of phospholipids in sera and cancer cells may explain their transport through cell membranes and significance of this mechanism in apoptosis.

Phospholipids (phosphoglycerides and sphingomyelins) are essential elements of cellular membranes. Phosphatidylcholine (PC) and sphingomyelin (SM) appear mainly in outer (exterior) leaflet of membrane. Phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) – are in inner leaflet. In plasma of healthy person PC, SM, LPC and PE prevail. PI and plasmalogens appear in plasma only in inconspicuous quantity. Cardiolipin (CL) is essential element of mitochondrial membranes. Sphingomyelin is the most known phospholipid in cancer disease as an important participant of signal transduction pathway.

In our investigation 31P NMR spectra were obtained from: sera, sera with added the sodium salt of cholic acid, methanol-chloroform extracts of phospholipids from: plasma, peripheral blood mononuclear cells (PBMC), bone marrow mononuclear cells (BMMC). Isopropanol-hexan extracts of phospholipids we used to extracted: peripheral blood erythrocytes (PBE), bone marrow erythrocytes (BME). Blood samples were collected by venous puncture after an overnight fast, for sera – 10 ml, for methanol chloroform extracts of phospholipid from plasma, PBMC, BMMC, PBE, BME 9 -15 ml. Cellular lipids were isolated from mononuclear cells Ficoll buffy coat centrifugation, and next underwent methanol-chlorophorm extraction. All spectra came from phospholipid extracts prepared from 60 x 106 cells for PBMC, BMMC and 5 x 109 for PBE, BME. Studies were carried out on AMX 300 Bruker and Avance III Bruker spectrometer 7.05 T.

In our preliminary studies we observed that 31P NMR spectra of normal serum consist of three peaks including a downfield peak due to Pi and two additional upfield field peaks from phospholipids PE+SM and PC. Spectra were performed in healthy volunteers, patients with acute leukemia, malignant lymphomas and multiple myeloma, at the time of diagnosis and repeated up to 13 times during chemotherapy. The sodium salt of cholic acid added to serum caused separation of three phospholipid peaks located upfield from inorganic phosphate. Contrary to the earlier studies, peaks from PE+SM and PC, and also a peak from LPC were observed. The above mentioned separation method had also been applied in investigations of patients with digestive tract tumors and with renal cell carcinoma. Changes in phospholipids in the 31P NMR spectra observed in these patients were primarily dependent on the advance of the disease. Long-term follow-up studies showed a good correlation between this 31P MRS evaluation of sera and the response of the disease to the therapy. At the time of diagnosis spectra showed strongly reduced peak areas and intensities from phospholipids (PC, LPC and PE + SM). During chemotherapy important changes in spectra

34

were observed: (1) in responding patients the spectral profile changed to resemble that of normal serum with increased peak intensities, (2) in non-responding individuals peak intensities were reduced. Spectra of patients suffering from acute leukemia or HD, who have achieved complete remission for 4-12 years did not differ from spectra of healthy volunteers. 31P NMR spectra can prove the presence of the residual leukemia in patients when the number of leukemic cells equals to 109 and is not detectable in laboratory tests of the blood and bone marrow. 31P NMR spectra can depict the function of the transplantated. It is possible to estimate the efficiency of cytostatics: e.g. DHAD & ARA-C as a first course treatment in patients with acute leukemia.

In our study 31P MRS spectra of normal extract of serum consist of six peaks due to phospholipids. Beside previously identified, i.e. PC, LPC, PE and SM, some new ones were observed: PI, CPLAS. 31P NMR spectra of phospholipid extracts from plasma. In patients with hematological cancers the values of peaks areas of PC, CPLAS, LPC, SM decreased. In some patients peaks from PI, PE were not observed. In responding patients the spectral profile changed to resemble that of normal extract. 31P MRS spectra of extract of mononuclear cells consist of 9 peaks due to phospholipids: PC, CPLAS, LPC, SM, PE+PI, PS, CL. The peak of LPC that has the most prognostic value in sera, in extract of mononuclear cells was observed only in some healthy volunteers. Important meaning for prognosis of disease course has got a presence of CPLAS (PAF). In the case of booth the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers. We focused on the significant difference in the integral intensities and phospholipid concentrations of SM and PS as well as CPLAS and PI+PE between the ALL and AML

groups.

The experiences already achieved pointed out that 31P spectra at present didn't allow for diagnosis of hematological disorders, but were of great importance in monitoring of therapy of the diseases under consideration. The changes of concentrations of phospholipids in sera and cells in hematological cancers are probably due to the increased uptake of phospholipid metabolites in proliferating blast cells, and their disturbed transport through cell membranes. It seems that SM and CPLAS (PAF) are of great importance in this process. It is very likely that observed reduction of the level of SM in blast cells’ extraction is due to activation of sphingomyelinase (SMnase) by for example tumor necrosis factor (TNF) or PAF. SMnase cleaves SM, generating choline phosphate and ceramide. The latter – among other things – mediates programmed cell death (apoptosis), induces differentiation and inhibits growth of leukemia cells. 31P MRS it may be useful method for understanding the meaning of phospholipids in mechanism of cancer’s proliferation. In this review of our study, it was shown how 31P MR spectroscopy can be applied for observations of changes of phospholipid concentrations in sera, plasma, PBMC BMMC, PBE, BME in patients with cancer diseases. This investigation points out that 31P MR spectra allow monitoring of phospholipid concentration in body fluid as well as in cells. Moreover, among acceptable various applications of 31P MRS, the possibility evaluating an advancement of disease, monitoring the treatment and forecasting resistance to the therapy seems to be the most promising. Simplicity of presented methods makes 31P MR spectroscopy a very useful tool for investigations of phospholipid metabolism.

35

PERFORMANCE OF DFT, SOPPA, SOPPA(CCSD) AND CCSD(T) METHODS IN PREDICTING NUCLEAR ISOTROPIC SHIELDINGS

IN THE COMPLETE BASIS SET LIMIT

Teobald Kupka1, Michał Stachów1, Marzena Nieradka1, Jakub Kaminsky2, and Tadeusz Pluta3

1University of Opole, Faculty of Chemistry, Opole, Poland; 2Department of Molecular

Spectroscopy, Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic; 3University of Silesia, Institute of Chemistry, Katowice, Poland

Convergence patterns and limiting values of isotropic nuclear magnetic shieldings

were studied for several small molecules (N2, CO, CO2, NH3, CH4, C2H2, C2H4, C2H6 and C6H6) in the Kohn-Sham limit. Individual results of calculations using dedicated families of Jensen’s basis sets (pcS-n and pcJ-n) and simple two-parameter formula were fitted toward the complete basis set limit (CBS) and ZPV correction applied. Several density functionals were used, and, for comparison purposes, calculations were performed using RHF, MP2, SOPPA, SOPPA(CCSD) methods and aug-cc-pVTZ-J basis set. Very demanding CCSD(T) calculations were performed in some cases, too. Finally, the CBS estimated results were critically compared with earlier reported literature data and experiment.

Literature 1. A. Auer, J. Gauss, J.F. Stanton, Quantitative prediction of gas-phase 13C nuclear magnetic shielding constants, J. Chem. Phys. 118, 10407 (2003). 2. T. Kupka, B. Ruscic and R. E. Botto, Toward Hartree-Fock- and Density Functional Complete Basis-Set Predicted NMR Parameters, J. Phys. Chem. A., 106, 10396 (2002). 3. T. Kupka and C. Lim, Polarization-Consistent vs Correlation-Consistent Basis Sets in Predicting Molecular and Spectroscopic Properties, J. Phys. Chem. A, 111, 1927 ( 2007). 4. T. Kupka, H2O, H2, HF, F2 and F2O nuclear magnetic shielding constants and indirect nuclear spin-spin coupling constants (SSCCs) in the BHandH/pcJ-n and BHandH/XZP Kohn-Sham limits, Magn. Reson. Chem., 47, 959 (2009).

36

THEORETICAL AND EXPERIMENTAL STUDIES ON CYTOSINE AND 5-FLUOROCYTOSINE

Teobald Kupka1, Mariana Spulber2, Mariana Pinteala2, Adrian Fifere2, Agnieszka Raniszewska1, and Malgorzata Broda1

1University of Opole, Faculty of Chemistry, Opole, Poland; 2 Institute of Macromolecular Chemistry Petru Poni, Iasi, Romania

Introduction of halogen atom into position 5 of DNA fragments affects their physico-chemical properties, including conformer stability, protonation states and hydrogen bonding properties. 5-fluoroderivatives are especially important and are widely used as drugs in cancer and fungal therapy. Such systems are very promising as controlled delivery systems in nanomedicine.

This paper discusses possibilities of theoretical methods in predicting structure, IR, Raman and NMR parameters of cytosine and 5-fluorocytosine in the gas phase and solution.

Literature

1. M. Spulber, M. Pinteala, A. Fifere, V. Harabagiu, B. C. Simionescu, Inclusion complexes of 5-flucytosine with β-cyclodextrin and hydroxypropyl-β-cyclodextrin: Characterization in aqueous solution and in solid state, J. Incl. Phenom. Macroc. Chem., 62 (2008) 117.

2. B. Blicharska and T. Kupka, Theoretical and Experimental NMR Studies on Uracil and 5-Fluorouracil, J. Mol. Struct., 613, (2000)153.

37

THEORETICAL CALCULATION OF NMR SHIELDINGS AND INDIRECT SPIN-SPIN COUPLING CONSTANTS

Teobald Kupka

1University of Opole, Faculty of Chemistry, Opole, Poland

Computational chemistry has been widely used to support assignment of experimental NMR spectra and for prediction of chemical structure-NMR spectra relation. The paper will discuss the role of various theoretical methods and basis set selection in obtaining meaningful NMR parameters without relaying on accidental error cancellation. The current limits of prediction for small and medium size atomic systems in the gas phase and solution will be addressed. Some ideas about applying theoretical methods to predict accurate NMR parameters of selected atoms within larger molecules, for example in active sites of enzymes, will be outlined, too. Literature 1. T. H. Dunning, Jr., J. Chem. Phys. 1989, 90, 1007. 2. Auer, J. Gauss, J. F. Stanton, J. Chem. Phys. 2003, 118, 10407. 3. U. Benedikt, A. A. Auer, F. Jensen, J. Chem. Phys. 2008, 129, art. no. 064111. 4. M. J. Frisch, et al., Gaussian 03, Revision E01,. Gaussian, Inc., Wallingford CT, 2004 (and recent Gaussian 09). 5. T. Helgaker, M. Jaszunski, M. Pecul, Prog. Nucl. Magn. Reson. 2008, 53, 249. 6. K. Dybiec, A. Gryff-Keller, Magn. Reson. Chem. 2009, 47, 63. 7. F. Jensen, J. Chem. Theor. Comput. 2008, 4, 719. 8. T. W. Keal, D. J. Tozer, T. Helgaker, Chem. Phys. Lett. 2004, 391, 374. 9. T. Kupka, B. Ruscic, R. E. Botto, J. Phys. Chem. A. 2002, 106, 10396. 10. T. Kupka, C. Lim, J. Phys. Chem. A. 2007, 111, 1927. 11. T. Kupka, Magn. Reson. Chem. 2008, 46, 851. 12. T. Kupka, Chem. Phys. Lett. 2008, 461, 33. 13. T. Kupka, Magn. Reson. Chem., 2009, 47, 210 14. T. Kupka, Magn. Reson. Chem. 2009, 47, 674. 15. T. Kupka, Magn. Reson. Chem. 2009, 47, 959.

38

CONFORMATIONAL ANALYSIS OF CYCLIC DYNORPHIN

ANALOGUES USING NMR DERIVED DATA

Kwasiborska Marias, Agnieszka Zieleniakb, Nowakowski Michała, Oleszczuk Martac, Wójcik Jaceka, Chung Nga N.d , Schiller Peter W.d, and Izdebski Janb

aLaboratory of Biological NMR, Institute of Biochemistry and Biophysics, PAS, Warszawa,

Poland; bPeptide Laboratory ,Warsaw University, Department of Chemistry, Warszawa, Poland; c Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada;

dLaboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, Canada

Previously we have reported results of our studies of cyclic dermorphin and deltorphin

analogues containing a carbonyl bridge [1-3]. We observed very high dual affinity for both µ and δ receptors of analogues containing the common (message) 1 - 4 sequence of those two native peptides. Elongation of the message sequence at the C-terminus to obtain peptides related to the full sequence of dermorphin [3] resulted in substantial changes of activity and selectivity for receptors. We found that added sequence effected selectivity of peptides and changed conformation of their N-terminal portions (message).

In this presentation we report biological activity, NMR and EDMC studies of new opioid peptide analogues, which contain modified enkephalin sequence (message) [4]: (1) D-Lys2,Lys5; (2) D-Lys2,Orn5; (3) D-Lys2,Dap5; (4) D-Orn2,Lys5; (5) D-Orn2,Orn5; (6) D-Orn2,Dab5; (7) D-Orn2,Dap5. In addition the C-terminal sequence of dynorphin (address) was added in each case, namely: -Arg6-Arg7-Ile8-Arg9-Pro10-Lys11-Leu12-Lys13.

The biological activity of peptides was measured with GPI/MVD tests.

NMR spectra of peptides 1, 3, 4 and 7 were measured at 25 °C on a UNITY500plus (Varian) spectrometer. TOCSY and gHSQC spectra were measured for assignment of all proton and carbon signals. ROESY experiments were performed in order to obtain distance restraints.

The conformational space of each peptide was explored using the EDMC method and NMR restraints were employed to select conformations present in the solution. Comparison of biological and conformational results allowed drawing SAR conclusions. Possible biologically active conformation was discussed.

References: [1] Filip K., Oleszczuk M., Pawlak D., Wójcik J, Chung N.N., Schiller P.W., Izdebski J. J.Peptide Sci., 9, 649-657 (2003). [2] Filip K., Oleszczuk M., Wójcik J., Chung N.N., Schiller P.W., Pawlak D., Zieleniak A., Parcińska A., Witkowska E., Izdebski J. J. Peptide Sci., 11, 347-352 (2005). [3] Witkowska E., Nowakowski M., Oleszczuk M., Filip K., Ciszewska M., Chung N.N., Schiller P.W., Izdebski J. J. Peptide Sci., 13, 519-528 (2007). [4] Pawlak D., Oleszczuk M., Wójcik J., Pachulska M., Chung N.N., Schiller P.W., Izdebski J. J. Peptide Sci., 7, 128-140 (2001).

39

DYNAMICS OF HYDROXYL DEUTERONS AND BONDED WATER MOLECULES IN NADY(0.8) ZEOLITE AS STUDIED BY MEANS

OF DEUTERON NMR SPECTROSCOPY

Z. T. Lalowicz1, G. Stoch1, A. Birczyński1, and M. Punkkinen2

1H. Niewodniczański Institute of Nuclear Physics of PAS, 31–342 Kraków, Poland; 2Wihuri Physical Laboratory, Department of Physics, University of Turku,

FI–20014 Turku, Finland

Deuteron spin–lattice relaxation and spectra were measured for NaDY(0.8) zeolite containing some heavy water. Two subsystems of deuterons with different mobility were disclosed at low temperatures. The corresponding relaxation rates differ by two orders of magnitude. Spectra exhibit different shapes related directly to a specific motional model. Hydroxyl deuterons perform incoherent tunneling along the hydrogen bond, then on increasing temperature jumps to excited states and over the barrier appear. Hydrogen bonded water molecules perform 180 rotational jumps about the twofold symmetry axis. Spectral amplitudes are consistent with the water content of 13 D2O molecules per unit cell. Above about 240K translational mobility becomes significant and finally water molecules fly across the free space of cages. It leads to a characteristic change in the slope of the relaxation rate temperature dependence. Similar features have been observed in the spin-lattice relaxation for D2 [1] and CD4 [2] in cages of various zeolites.

References:

[1] J. S. Blicharski, A. Gutsze, A. M. Korzeniowska, Z. T. Lalowicz, and Z. Olejniczak, Appl. Magn. Reson. 27, 183 (2004).

[2] A. Birczyński, M. Punkkinen, A. M . Szymocha, and Z. T. Lalowicz, J. Chem. Phys. 127, 204714 (2007).

40

SPECTRAL DENSITY FUNCTIONS OF COMPLEX MOLECULAR MOTIONS

Lidia Latanowicza and Zofia Gdaniecb

a Faculty of Biological Sciences, University of Zielona Góra, Poland; bInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland

The fundamental claim of Woessner “Although the two types of motion are

independent, their contributions to relaxation are not. If the interaction Hamiltonian is modulated by series of independent stochastic processes, the total correlation function of a complex motion should be calculated” is the basis of the calculations of the total spectral density of complex motion.

The motion of spin pair distanced at Ris in liquid state is complex as it consists of the isotropic (anisotropic) overall motion and the internal motion of molecular groups. The NMR order parameter is calculated for the case of simultaneous changes in the radial and angular parts of dipolar Hamiltonian [1]. The radial motion takes place when only one spin in the spin pair is involved in the motion. Then Ris distance undergoes changes simultaneously with the jump angles Θis. The effect of the activation parameters of internal motions (known from the solid state study) on measured T1 relaxation in the liquid state temperature regime will be shown. The range of applicability of the model - free approach Lipari and Szabo in the study of molecular dynamics in solutions is considered. The order parameter, S2(model-free), concerns the angular amplitude of fast internal motion and it is defined as

) motion isotropic,(ωmis)/J motion c,(ωm

isJ2S omplexfreeel =− )(mod in the temperature regime of the

maximum of the function 2

isoτ2ω1

iso2τ

+. The value of S2 (model-free ) is temperature dependent

because the correlation times of internal τn and isotropic motions τiso are temperature dependent [2]. The influence of the temperature dependencies of spectral densities overlap on the relation between S2(model-free) and the true value of the order parameter S2 is analyzed. The temperature regime of the equality S2(model-free) = S2 is estimated.

0 5 10 15 20

0,2

0,4

0,6

0,8

1000/T (K-1)

J2 (ωi+ω

s) [s

-1]

(a)(1)(4)(iso)

0 2 4 6 8

0,10,20,30,40,50,60,70,80,91,01,1

(5)

(2)

(3)(4)

(1)

S2 = cos2(109.40)

1000/T (K-1)

OR

DE

R P

AR

AMET

ER

S

2 (mod

el -

free)(b)

Fig.1 The illustration of the reductions in the spectral densities in the regime of the maximum of the function

2isoτ2ω1

iso2τ

+ under the influence of internal motion. Exemplary case of the complex motion (C3 + Ciso); solid

lines – the temperature dependencies of spectral density (a) where (ωs = 2π×75.432 MHz, ωi = 2π×300 MHz), and order parameter S2 (model - free) (b). The motional parameters chosen are: Ris = 0.109 nm, βis = 70.310

(the angle between the Ris and C3 rotation axis), s13108.3 −⋅=30τ , E3 = 2.9 kJ/mol (curve (1)), 4.2 kJ/mol

(curve (2)), 6.3 kJ/mol (curve (3)) , 8.4 kJ/mol (4), 12.6 kJ/mol (curve (5)), s14106.1 −⋅=iso0τ , Eiso = 20.5

kJ/mol (iso). The spectral densities for the single motion C3 and isotropic overall motion are shown by pointed and dashed lines respectively. The dashed arrow indicates room temperature.

41

The spectral density of a single motion which is Ciso goes through a maximum in the temperature dependence. Such a maximum is split into two maximums for the spectral density of Caniso motion. This split is due to the fact that the D⊥ and D⎢⎢ rotational diffusion coefficients are dominant in different temperature regimes because of the different rate of these motions. D⎢⎢ representing faster motion dominates at lower temperatures. The high splitting is observed when the activation parameters D⊥ and D⎢⎢ differ significantly (curve (1) in Figure 2). Curve (1) represents the high and curve (3) represents small anisotropy. Curve (3) is identical with curve (iso) –pointed line, representing the case D⊥ = D⎢⎢. We can conclude that anisotropic motion is impossible to be recognized in the liquid state temperature regime, because of the overlapping of the spectral densities of motions represented by D⊥ and D⎢⎢ rotational coefficients. This effect is similar to that of the overlap of spectral densities of not well-separated isotropic overall and internal motions.

Fig. 2. Exemplary temperature dependencies of the spectral densities )(0

siisJ ωω − where (ωs = 2π×75.432 MHz, ωi = 2π×300 MHz) and heteronuclear T1s temperature dependences for a spin pair (Ris = 0.109 nm) which performs a Caniso motion. The motional parameters chosen are: αis = 300 (the angle between main axis of the molecule and Ris ),

11300 105 −⊥ ×== sDD II , E⊥ = 24.3 kJ/mol, EII = 24.3 kJ/mol (the pointed curve (iso)), EII

= 10.5 kJ/mol (solid curve (1)), EII = 16.8 kJ/mol (solid curve (2)), EII = 21.8 kJ/mol (solid curve (3)). The curve (iso) is depicted by open circles to distinguish it from curve (3). The dashed arrow indicates room temperature.

The proton spin pairs of the methyl group are known to perform a complex motion being a resultant of three components involving mass transportation over and through the potential barrier [3]. They are characterised by the correlation times 3τ and Tτ of the jumps over the barrier and tunnel jumps in the threefold potential of the methyl group and isoτ the correlation time of isotropic rotation. The recently derived equations for the tunnelling rate constant according to Schrödinger are taken into account in the calculations of the spectral densities of tunnelling jumps. References: 1. L. Latanowicz, Z. Gdaniec, Mol. Phys. 107 (2009) 1563 – 1576. 2. L. Latanowicz, Z. Gdaniec, J. Phys. Chem. submitted 3. L. Latanowicz, W. Medycki, R. Jakubas, Solid State NMR, accepted

0 5 10 15 20 25 30

50

100

150

200

250

300

350

400

450

1000/T (K-1)

J0 (ωi-ω

s) [s

-1]

(3)

(1)

(2)

(iso)

0 5 10 15 20 25 30

0,01

0,1(3)

(2)

(1)

(iso)

1000/T (K-1)

T 1s [s

]

0 10 20 30 40 50 60

0,1

1

#3

#2 90 MHz

1000/T (K-1)

PR

OTO

N T

1 (s) #1

Fig. 3 Exemplary case of the proton T1 for the complex motion of methyl group (C3 (classical) +C3(tunneling jumps) + Ciso) - #1, (C3 (classical) + Ciso) - #2 and single motion Ciso - #3. ωi = 2π×90 MHz, The motional parameters chosen are: Ris = 0.178 nm, ωT = 2π×3 MHz (tunnel splitting),

s12101 −⋅=30τ , s14107 −⋅=iso

0τ , s8108 −⋅=tu0τ ,

E3 = 3.3 kJ/mol, Eiso = 20.9 kJ/mol,

This paper is dedicated to the memory of NMR relaxation specialist, researcher Donald Edward Woessner (1930 – 2008). 42

SOLID STATE STUDIES OF CYCLODEXTRIN COMPLEXES WITH ADAMANTANE AND AMANTADINE USING 13C AND 15N

CP/MAS NMR

Agnieszka Lis-Cieplak and Wacław Kołodziejski

Department of Inorganic and Analytical Chemistry, Faculty of Pharmacy, Medical University

of Warsaw, Warsaw, Poland

Cyclodextrins (cyclomaltooligosaccharides) are macrocyclic oligosugars capable of forming inclusion complexes with small hydrophobic molecules, thus improving their water solubility [1,2]. Beta-cyclodextrin (β-CD) host is well suited for many guest drug molecules and this prompts various pharmaceutical applications, especially in drug formulation and delivery. The complexation with β-CD significantly modifies drug solubility, bioavailability and stability [3,4]. Therefore, the β-CD/drug complexes seem worthy of careful studies, including their solid forms used for preparing tablets. Cyclodextrin complexes with various pharmaceutical compounds were widely investigated using solution and solid-state NMR [1,3,4]. Amantadine (1-aminoadamantane) is used as an antiviral and antiparkinsonian drug. We report on the β-CD/amantadine complex, which has been investigated under magic-angle spinning (MAS) using solid-state 13C and 15N NMR spectroscopy with cross-polarization (CP) from protons. For comparison, we have also studied the β-CD/adamantane complex and both uncomplexed guest compounds. The NMR spectra supplied us with structural details and provided interesting information on molecular dynamics. The latter subject has been successfully studied using kinetics of CP [5]. CP is considerably sensitive to internuclear distances and to mobility of molecules and/or their functional groups in the solid state. The CP kinetics has been monitored using variable-contact-time measurements and analyzed in terms of known CP models [5].

The 1H → 13C CP kinetics has indicated that the complexation affects molecular arrangement and interactions in the solid state, decreases molecular mobility of the guest molecules, while the host macrocycle becomes less rigid in the macromolecular complex. The study is to be continued with other drugs containing the adamantyl functional group. References: [1] H. Dodziuk, Cyclodextrins and Their Complexes, Wiley-VCH, Weinheim (2006) [2] W. Saenger, J. Jacob, K. Gessler, T. Steiner, D. Hoffmann, H. Sanbe, K. Koizumi, S.M.

Smith, T. Takaha, Chem. Rev. 98, 1787-1802 (1998) [3] U. Holzgrabe, I. Wawer, B. Diehl, NMR Spectroscopy in Drug Development and Analysis, Wiley-VCH, Weinheim (1999) [4] K. Uekama, F. Hirayama, T. Irie, Chem. Rev. 98, 2045-2076 (1998) [5] W. Kołodziejski, J. Klinowski, Chem. Rev. 102, 613-628 (2002)

43

MOLECULAR DYNAMICS AND STRUCTURE OF MIKTOARM STAR BLOCK COPOLYMERS BASED ON POLY(BUTYL ACRYLATE)

AND POLY(ETHYLENE OXIDE)

Monika Makrocka-Rydzyka, Aleksandra Wypycha, Mariusz Jancelewicza, Stefan Jurgaa*, and Krzysztof Matyjaszewski b.

a Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University,

Poznań, Poland; bDepartment of Chemistry, Carnegie Mellon University,Pittsburgh, Pennsylvania, USA

The strategy in modern chemistry is to made large oligomers from relatively simple building blocks. It follows a way of the nature of performing molecules by joining small units together through heteroatom links. Use of different types of chains attached to a central core leads to combinatorial with remarkable modularity and diversity. This class of polymers embraces miktoarm star copolymers, i.e. star-shaped compound containing arms with different chemical compositions. They were synthesized by means of the atom transfer radical polymerization technique [1]. Star-shaped copolymers have applications in drug delivery, diagnostic assays, nanopatterned structures and photonics [2]. The investigated miktoarm star copolymers are composed of poly(butyl acrylate) - polyBA and poly(ethylene oxide) - polyEO chains connected to a central core. The molar ratio of polyBA/polyEO was: 0.80/0.20, 0.50/0.50, 0.80/0.20. The Broadband Dielectric Spectroscopy (BDS), performed in wide temperature range allowed to detect in polyBA/polyEO 0.20/0.80 and polyBA/polyEO 0.50/0.50 two relaxation processes: α relaxation process - assigned to segmental chain motions of polymer backbone and αc relaxation - coming from chain mobility in amorphous phase, constrained between crystallites. The BDS studies for polyBA/polyEO 0.80/0.20 do not reveal αc-process probably due to high amount of amorphous polyBA, which prevents the crystallization process. Fast Field Cycling NMR studies (FFC) performed for polyBA/polyEO 0.50/0.50 below melting temperature (Tm) confirm dynamics of polymer chains in confined space, revealing dispersion of spin-lattice relaxation times obeying power law T1∝ ω0.75 [3]. However above Tm the frequency dependence of T1 is described by power laws: T1∝ ω0.5 and T1∝ ω0.25 at high and low frequencies, respectively. They reflect chain dynamics, which can be explained on the basis of the renormalized Rouse model in terms of high-mode and low-mode number limit [4]. Acknowledgements: This work is supported by 6th Framework Programme under SoftComp Grant No 502235-2 and research grant No N N202 128536 (Poland). A. Wypych acknowledges the Ministry of Science and Higher Education in Poland for a Postdoctoral Fellowship (POL-POSTDOC III).

References: [1] Haifeng Gao and Krzysztof Matyjaszewski; Macromolecules, 2008, 41, 4250-4257. [2] John E. Moses and Adam D. Moorhouse, Chem. Soc. Rev., 2007, 36, 1249–1262. [3] Reiner Kimmich and Esteban Anoardo, Prog. NMR Spectrosc. 2004, 44, 257-320. [4] Nail F. Fatkullin and Reiner Kimmich; J. Chem. Phys. 1994, 101, 822-832.

44

MOLECULAR DYNAMICS AND NUCLEAR RELAXATION PROCESSES OF PLANAR CATIONS WITH PSEUDO 5-FOLD

SYMMETRY AXES: IMIDAZOLIUM AND PYRAZOLIUM COMPOUNDS

W. Medycki1, D. Kruk2, R. Jakubas3, and J. JadŜyn1

1Institute of Molecular Physics, Polish Academy of Sciences, Poznań, Poland;

2Institute of Physics, Jagiellonian University, Kraków, Poland; 3Faculty of Chemistry, University of Wrocław, Wrocław, Poland

Imidazole molecules are the smallest ones possessing properties favorable for creating

new ferrolectric materials and therefore their dynamics attracts considerable attention. In this work we study molecular dynamics of selected imidazolium and pyrazolium

compounds: Im3Sb2Br9, Im6Bi2Br18, Im5Bi2Cl11, Im5Sb2Br11 and Pl4[Bi2Br10]x2H2O, by means of NMR relaxation experiments.

In some of the previous studies [1] the relaxation features of these systems have been attributed to two small angle libration modes, at lower temperatures, and to regular C5 jumps about the axis perpendicular to the centre of the cation plane, at higher temperatures.

At present we attempt to explain the complex temperature dependencies of the proton relaxation rates as a result of the quadrupolar interactions leading to efficient relaxation channels for the nuclei possessing quadrupolar moments [2]. The quadrupolar relaxation mediates, in turn, the relaxation dynamics of protons via mutual dipole-dipole couplings.

This work has been financed by Polish Ministry of Science and Education, grant No N N202 172135 References:

1. A. Piecha, A. Białońska, R. Jakubas, W. Medycki, Solid State Sciences. 10 (2008) 1469-1479; 2. A. Piecha, R. Jakubas, A. Pietraszko, J. Baran, W. Medycki, D. Kruk, Journal of Solid State Chemistry

182 (2009) 2949–2960.

45

THEORY OF SOLID STATE DYNAMIC NUCLEAR POLARIZATION

Artur Mielczarek1, 2, and Danuta Kruk2

1 Instytut Fizyki Jądrowej PAN, Kraków, Poland; 2 Institute of Physics, Jagiellonian University, Krakow, Poland

Solid-state dynamic nuclear polarization experiments are commonly performed by

irradiating a sample that contains dilute paramagnetic species with a saturating microwave field oscillating with, or close to, the electron Larmor frequency. Thereby the polarization of the nucleai in the sample is enhanced above the equilibrium value, by a factor in the ideal case given by the electronic divided by the nuclear gyromagnetic ratio. To achieve the enhancement of the nuclear polarization an effective coupling between the nuclear and the electronic spins is needed. The interaction leads to eigenfunctions of the electronic – nuclear spin system being a mixture of ‘ordinary’ Zeeman states. Populations of the individual energy levels evolve in time following a set of relaxation equations. The relaxation coefficients, which connect the energy levels, depend on the alternating microwave field and the absorption spectrum (ESR spectrum) of the electron spin involved. The ESR lineshape function, is crucial for the dynamic nuclear polarization process. The amplitude of an ESR spectrum at a given frequency is the fraction of electron spins with transition frequency equal to that frequency. Therefore the ESR lineshape function gives “weigh factors” for the transition probabilities (relaxation coefficients). The ESR spectrum is determined by many parameters and has to be calculated independently starting from an electron spin Hamiltonian appropriate for the considered case. During the evolution there is a time point at which the momentary populations of the energy levels are particularly favorable for the nuclear spin leading to an enhanced nuclear polarization. The enhancement does not last forever; after some time all populations are equilibrated. This short time period of enhanced nuclear polarization is very profitable in NMR investigations of biological systems. We present a perturbative description of Dynamic Nuclear Polarization (DNP) in solids. It is very common in the literature [eg. 1,2] to distinguish between DNP mechanisms like ‘solid effect’ or ‘thermal mixing’. This long standing tradition is somewhat confusing because it suggests a different origin of these processes, while the difference lays, in fact, in the strength of the electron – nuclear spin dipole – dipole coupling. Here we propose for a first time (according to our knowledge) an approach based on a perturbation solution of the Liouville-von Neumann equation, which does not require any ‘a priori’ presumptions regarding the interaction strengths. The final DNP enhancement factor is obtained as a solution of population equations including transition probabilities resulting from the alternating magnetic field and electron- and nuclear spin relaxation. The DNP factor is linked to a corresponding Electron Spin Resonance (ESR) lineshape, which is simultaneously evaluated within the same set of parameters. References: [1] A. Abragam and M. Goldman Rep. Prog. Phys., 41 395-467 (1978) [2] T. Maly, G. T. Debelouchina, V. S. Bajaj, K.-N., Hu, C.-G. Joo, M. L. Mak-Jurkauskas, J. R. Sirigiri, P. C. A. van der Wel, J. Herzfeld, R. J. Temkin and R. G. Griffin J. Chem. Phys. 128(5) (2008) This work has been financed by Polish Ministry of Science and Education, grant No N N202 105936

46

FIELD DEPENDENT RELAXATION PROCESSES IN MULTISPIN SYSTEMS

Agnieszka Milewska and Danuta Kruk

Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Krakow, Poland

This work is concerned with several aspects of field dependent relaxation processes in

multi-spin systems containing unlike nuclear spins (like poly-alcohols) and nuclear and

electron spins (like radicals and bi-radicals). Multiexponentiality of relaxation processes in

such systems is investigated in detail. The exponentiality problem is a very important issue

for a proper understanding and description of relaxation effects limiting the efficiency of

polarization transfer between electron and nuclear spins. Nuclear spin relaxation resulted from

electron spin interaction for selected radicals is analyzed in detail.

This work has been financed by Polish Ministry of Science and Education, grant No N N202 105936

47

THREE-DIMENSIONAL NMR SPECTROSCOPY OF ORGANIC MOLECULES

Maria Misiak and Wiktor Koźmiński

Faculty of Chemistry, University of Warsaw,

Warsaw, Poland

Multidimensional NMR Spectroscopy can be regarded as a powerful technique used for studies of molecular structure and dynamics of complex organic compounds.

Here we present the application of 3D NMR experiments, which are based on random sampling of the evolution time space followed by Multidimensional Fourier Transform (MFT). This approach, applied at first to the strychnine molecule, enables one to acquire 3D spectra in reasonable experimental time and allows retaining high resolution in indirectly detected domains.

We also show that by the application of 3D NMR experiments we were able to do the complete 1H and 13C spectral assignment of natural abundance prenol-10, what was impossible earlier using 1D and 2D techniques because of the complexity of the spectra.

Furthermore we show here an application of 3D NMR experiments for measurement of heteronuclear coupling constants of organic compounds yielding complex spectra. The interpretation of 3D HSQC-TOCSY spectra with E.COSY-type multiplets allowed us to evaluate heteronuclear coupling constants of strychnine with high accuracy, what is difficult employing 2D methods owing to signal overlap and impossible using conventionally recorded 3D NMR spectra in the reasonable time of an overnight experiment. References: [1] Kazimierczuk K, Koźmiński W, Zhukov I, J. Magn. Reson. 179, 323 (2006) [2] Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I. J. Biomol. NMR. 36, 157 (2006) [3] Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I. J. Magn. Reson. 188, 344 (2007) [4] Misiak M, Koźmiński W, Magn. Reson. Chem, 45, 171 (2007) [5] Kazimierczuk K, Zawadzka A, Koźmiński W. J. Magn. Reson. 192, 123 (2008) [6] Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I. J. Am. Chem. Soc. 130, (16) 5404 (2008) [7] Misiak M, Koźmiński W, Magn. Reson. Chem. 47, 205 (2009) [8] Misiak M, Koźmiński W, Kwasiborska M, Wójcik J, Ciepichal E, Swiezewska E, Magn. Reson. Chem., 47, 825 (2009)

48

APPLICATION OF MR MICROSCOPY FOR ASSESSMENT OF HYDRATION PROCESSES IN HPMC BASED MATRIX TABLETS

Anna Młynarczyk1, Marco L.H. Gruwel2, Piotr Kulinowski1, Krzysztof Jasiński1, Przemysław DoroŜyński3, Bogusław Tomanek2,1, and Władysław P. Węglarz1

1 Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, PAN, Kraków, Poland; 2 National Research Council of Canada, Institute for Biodiagnostics, Winnipeg, Canada; 3 Department of Pharmaceutical Technology and Biopharmaceutics, UJ, Kraków, Poland.

The study of tablets dissolution is needed to ensure efficient and timely drug delivery. In this work MR microscopy was applied for study of water mobility and concentration in tablets, used for drug delivery [1,2]. The tablets made of Hydroxypropylmethylcellulose-HPMC with and without addition of a drug substance of different solubility were tested (Tab.1.).

Tab.1. Tablet formulation with abbreviations used in text HPMC-1 Metolose60SH10000cP (100%)

HPMC-1 + KT Metolose60SH10000cP + Ketoprofen (50:50%) HPMC-1 + LD Metolose60SH10000cP + L-dopa (50:50%)

HPMC-2 Metolose65SH400cP (100%) HPMC-2 + KT Metolose65SH400cP + Ketoprofen (50:50%) HPMC-2 +LD Metolose65SH400cP + L-dopa (50:50%)

Experiments were performed on a 11.7T vertical bore magnet (Oxford Instruments U.K.) equipped with a 72mm ID gradient set (Magnex, UK) and an Avance console (Bruker,Germany). MR images were acquired at 22.0±0.5°C using a MSME sequence with a FOV=15x15mm, matrix size 256x256, TR/TE=4000/6,5ms, NEX=2, total scan time 30min. T2 and proton density calculations were carried out using Matlab (The MathWorks, Inc.).

Two-dimensional T2 and proton density (PD) maps were obtained. T2 maps for HPMC-1, HPMC-1+LD and HPMC-1+KT are shown in Fig.1. Histograms of these maps at 120 minutes of hydration are shown in Fig.2.

120 min

60 min

30 min

180 min A

120 min 30 min

60 min

180 min B

120 min 30 min

60 min

180 min C

Fig.1. Fragments of T2 maps at 30, 60, 120, 180 minutes: A) HPMC-1, B) HPMC-1+LD, C) HPMC-1+KT .

Fig.2. T2 map histograms at 120 minutes of hydration

49

Three modes corresponding to different physicochemical properties of polymer-water-drug system are observed. Spatial distribution of T2 and proton density along a tablet radius were also analyzed.

Different mechanisms of tablets hydration were observed due to composition of the formulations. The experiment has shown that the phenomena occurring during drug dissolution are closely related to the properties of applied substances. It is especially important for the research and the development studies leading towards more effective drug delivery systems. REFERENCES [1] C.Melia, et al, PSTT, 1998, 1(1): 32-39. [2] M.Vlachou, et al, Polym. Adv. Technol., 2004, 15:683-689. [3] S.Silva, et al,Journal of Colloid and Interface Science 2008, 327: 333-340. [4] G.Bajwa, et al, Polymer 2009, 50: 4571-4576.

50

3D NMR-BASED STRUCTURE OF apo-S100A1 HUMAN PROTEIN

Michał Nowakowski1, Łukasz Jaremko1,2, Igor Zhukov1,3, Agnieszka Belczyk1, Andrzej Bierzyński1, and Andrzej Ejchart1

1 Institute of Biochemistry and Biophysic, Polish Academy of Sciences, Warszawa, Poland; 2 College of Inter-Faculty Individual Studies In Mathematics and Natural Sciences, Warsaw

University, Warszawa, Poland; 3 Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia

S100 is a multigenic family of calcium-modulated proteins of the EF-hand superfamily implicated in intracellular and extracellular regulatory activities. Within cells, most S100 proteins exist in the form of antiparallely packed homodimers stabilized by the nonbonding interactions. It is widely accepted that proteins from S100 family are signalling proteins. S100A1 contains 93 residues per subunit. It is stabilized by noncovalent interactions at its dimer interface; each subunit contains two calcium binding loops. Using a number of multinuclear 3D NMR techniques applied to the sample of 13C,15N double labeled apo-S100A1 human protein measured at 11.7 T, 16.4 T and 18.8 T we have assigned chemical shifts of almost all backbone and side chain nuclei. Results were deposited in Biological Magnetic Resonance Bank [1] (entry number 16360). Secondary structure elements were predicted on the basis of 1H, 13C and 15N chemical shifts. Program PREDITOR [2] was applied to our chemical shift data confirming, as expected, existence of four α-helical structures in each subunit. Preliminary low resolution 3D structure of apo-S100 monomer was predicted using CS23D [3] program on the basis of chemical shifts and was very close to our expectations. RMSD calculated in comparison to rat monomer structure aligned by heavy atoms for residues not interacting with second subunit is 1.52 Å with prediction taking closest homologues from PDB into account, while it is 2.5 Å when alligned by all heavy atoms respectively.

Figure: Structure of human apo-S100A1 monomer calculated with CS23D (on the left), structure

of rat apo-S100A1 monomer (on the right).

51

This chemical shift based structural information was combined with distance constrains derived from 13C and/or 15N edited NOESY spectra and used to calculate 3D structure of apo-S100A1 human protein with CNS package [4, 5] and CYANA [6,7]. Calculated structure was compared with the structures previously determined for rat apo-S100A1 protein [8] and bovine apo-S100A1 protein modified by disulfide bond formation with β-mercaptoethanol [9]. Acknowledgment This work was supported by a grant N301 031234 from the Polish Ministry of Science and Higher Education References [1] http://www.bmrb.wisc.edu [2] M.V. Berjanskii, S. Neal,D.S. Wishart, Nucleic Acids Res. 2006, 34 [3] D.S. Wishart, D.Arndt, M. Berjanskii, P. Tang, J. Zhou, G. Lin, Nucleic Acids Res. 2008,36 [4] A.T. Brunger, P.D. Adams, G.M. Clore, P.Gros, R.W. Grosse-Kunstleve, J.-S. Jiang, J.

Kuszewski, N. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Acta Cryst. 1998, D54, 905-921

[5] A.T. Brunger, Nature Protocols 2007, 2, 2728-2733 [6] P. Güntert, C. Mumenthaler, and K Wüthrich J. Mol. Biol. 1997, 273, 283-298 [7] P. Güntert, W. Braun, K Wüthrich J. Mol. Biol. 1991, 217, 517-530 [8] R.R. Rustandi, D.M. Baldisseri, K.G. Inman, P. Nizner, S.M. Hamilto, A. Landar, A.

Landar, D.B. Zimmer and D.J. Webber, Biochemistry 2002, 41(3), 788-796 [9] Zhukov I, Ejchart A, Bierzyński A, Biochemistry 2008 , 47, 640-650

52

TOWARD CHIRAL CRYSTALS OF BENZODIAZACORONANDS

Katarzyna Nowicka1, Agata Jeziorna1, Adam Sobczuk2, Janusz Jurczak2, Grzegorz D. Bujacz3, Anna Bujacz3, Włodzimierz Ciesielski1, and Marek J. Potrzebowski1

1Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Łódź, Poland; 2Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland;

3Institute of Technical Biochemistry, Technical University of Łódź, Poland

In this work we present the application of solid state (SS) NMR spectroscopy and XRD in structural studies of inclusion complexes. On the course of our study we have found that benzodiazacoronands (host molecules) are able to form molecular complexes with different guests. The main aim of this project is to develop the methodology of searching of inclusion complexes in solid state, employing SS NMR and XRD as complementary tools. 13C CP/MAS spectra of benzodiazacoronand with different guest molecules (acetone, toluene, methanol) in the crystal lattice are presented below.

53

OPTIMIZATION METHOD FOR ADIABATIC TAGGING PULSES FOR ARTERIAL SPIN LABELING

Wojciech Obrębski, Piotr Bogorodzki, and Ewa Piatkowska-Janko

Institute of Radioelectronics, Faculty of Electronics and Information Technology at the Warsaw University of Technology, Warsaw, Poland

Introduction

Magnetically marked water used in MRI imaging allows us to obtain perfusion images as well as measuring cerebral blood flow (CBF) with comparable spatial resolution and sensitivity to Blood Oxygenation Level dependent (BOLD) imaging. In this work we deal with a technique called arterial spin labeling (ASL). ASL is a safe imaging technique working without contrast agents allowing thus multiple and safe repetitions. A major drawbacks of ASL are limited time resolution (due to the additional time required to label the blood) and relatively low signal to noise ratio. In order to obtain acceptable image quality a increased number of repetitions is required exposing patients on additional dose of SAR. The aim of this work was to propose a new approach to optimization of adiabatic pulse used for blood tagging. Optimized with the new method pulses use less power for labeling, so the tagging can be carried out continuously, regardless of the scanner activity.

Subjects and Methods The proposed method is based on assumption, that pulse which efficiency is optimized is non-selective and adiabtic factor should by constant during pulse execution. Optimized with the new methodology pulses will be compared with the Silver−Joseph−Hoult (SJH, hyperbolic secant) impulses either by computer simulation or experimental measurements on a Brucker BNT1000 scanner. Proposed pulses were generated by home made pulse programmer based on FPGA (Xilinx Spartan3 XC3S200) and direct digital synthesis (DDS) chips (Analog Devices AD9851).

Results and Conclusions

The optimized pulse showed 60% increase in efficiency in the computer simulations. In consists of two simple, easy to implement discrete functions. The designed coil is expected to allow a considerable reduction in the power required for the imaging and realize truly continuous ASL

54

NON-DEBYE RELAXATION AND TEMPERATURE DEPENDENCE

OF THE SECOND MOMENT OF NMR LINE

Marcin Olszewski and Nikolaj Sergeev

Institute of Physics, University of Szczecin, Poland

The temperature dependence of the second moment 2M of NMR line is determined by expression [1]

∫δω

δω−

ωωπ

= dJTM )(1

)( 02 , (1)

where the spectral density )(0 ωJ is the Fourier transform of the dipolar correlation function

)(th ( 0>t ) [1]

dttωithωJ ∫∞

∞−

⋅= )exp()(Re)(0 . (2)

Usually the dipolar correlation function is selected as exponential Debye function ( )Cτthth /exp)0()( −= , where Cτ is the correlation time described the relaxation of the

dipolar correlation function. The Fourier transform of this function gives the Debye (or Bloembergen-Purcell-Pound) form of the spectral density )(0 ωJ [1]

( )201

)0(2)(

C

C

τω

τhωJ

⋅+⋅= . (3)

However the non-exponential relaxation processes are often observed in the different range of physics (see [2-5] and references there). The non-exponential relaxations give the spectral densities )(0 ωJ differed from the Debye form. At present it is well known the

several spectral densities functions described the non-Debye relaxation [2-5]. It have been used frequently the Cole-Cole function; the Cole-Davidson function; and Havriliak-Negami function [3-5]. These functions may be wrote by one expression [3]

( ) ( )( ) ( )

( ) ( ) ( )[ ] 2/202/cos21

2/cos1

2/sinarctansin)0(2

),,(εδ

Cδ

C

δ

C

δ

C

ωτδπωτω

δπωτ

δπωτεh

δεωJ++⋅

+⋅

= , δεδ /1,10 ≤≤< . (4)

Spectral density (4) coincides with Debye function (3) if 1== δε [3]. In the present communication it will be discussed the temperature dependence of the

second moment of NMR line for the case of non-Debye (non-exponential) relaxation. The temperature dependences of the spin-lattice relaxation time have been considered in [3.] References: [1] A.Abragam, The Principles of Nuclear Magnetism (Oxford Univ.Press, Oxford, 1961). [2] J.C.Phillips, Streched exponential relaxation in molecular and electronic glasses, Report Progress Physics, 59 (1996) 1133-1207. [3] P.A.Beckmann, Spectral densities and Nuclear Spin Relaxation in solids, Physics Reports, 171 (1988) 86-128. [4] W.T.Coffey, Dielectric relaxation: an overview, Journal of Molecular Liquids, 114 (2004) 5-25. [5] W.Dieterich, P.Maass, Non-Debye relaxations in disordered ionic solids, Chemical Physics, 284 (2002) 439-467.

55

ANALYSIS OF BOLD SIGNAL IN THE BRAINSTEM DURING RESTING STATE AND AFTER CONTINUOUS SOUND ACTIVATION

Paulina Palowska1, Michalina Ryń2, Uwe Klose2, and Zofia Drzazga1

1Department of Medical Physics – University of Silesia, Katowice, Poland;

2 Department of Diagnostic an Interventional Neuroradiology – University Hospital Tübingen, Tübingen, Germany

Introduction

Functional magnetic resonance imaging (fMRI) is a powerful method which can detect discrete areas of slight perfusion changes within the brain resulting from neural activity. Visualization of functional magnetic resonance imaging activation of subcortical structures remains challenging because of the cardiac – related pulsatile movement of both the brainstem and the cerebrospinal fluid and involved special scanning, pre – and postprocessing techniques.

The purpose of this study is to develop fMRI paradigms. Activations in brainstem nuclei were investigated during continuous sound (continuous stimulus) and resting state measurements in correlation analysis. By using special technique like cardiac gatting fMRI it was possible to obtain activations only in few cases. That why it was used additional regressor in SPM analysis – signal changes from white matter to obtain better results of activations in brainstem. Materials and Methods

Subjects: A total of ten right handed volunteers (aged 26.8 ± 6.7) were examined. They were all free of neurological disease and did not report a history of hearing problems.

Data acquisition: In this examinations a whole body scanner (3T Trio Tim, Siemens Erlangen, Germany) was used. Signals were detected by BOLD differences using T2* - weighted EPI technique. A total of 153 data sets were acquired every second heart beat – TR depend on cardiac rhythm and TE = 30ms or 34ms (conditional on slice thickness). The volumes consisted of 10 slices with slice thickness 2.5 mm, field of view (FoV) 192mm and base resolution 64 or with slice thickness 1.7 mm, field of view (FoV) 192mm and base resolution 96. After functional data T1 – weighted spin echo anatomical scans were acquired with parameters: TR = 2300ms, TE = 3.03ms, flip angle = 8 degrees, slice thickness = 1mm, FoV = 256mm, voxel size 1.0x1.0x1.0mm and base resolution 256.

Data analysis: All data were analyzed and modified by the software package SPM5 and home – written routines in MatLab. Measured variable repetition times were used in fMRI model specification as a regressor (Fig. 1. a). Since TRs as regressor was insufficient it was necessity to used signal changes from white matter as additional regressor (Fig. 1. b) what considerably improved quality of images.

pixel coordinates

pixel coordinates

Correlation map

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60

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Fig 1. Correlation analysis: a) with TR as regressor b) with TR and white matter.

a) b)

56

Results and Discussion The positions of auditory cortex were found and the analysis of correlation was

obtained. The auditory cortex and brainstem nuclei were identified in correlation analysis with the threshold of correlation coefficient 0.5. Positions of the brainstem were correlated with the auditory coordinates with the highest value of T (from block design experiments). Numbers of activated localizations in brainstem nuclei which were found in correlation analysis during continuous sound and resting state are shown in Table 1. Correlation map for continuous sound experiment and time course from auditory cortex (left and right side) and inferior colliculi (right) are demonstrated in Fig. 2.

CONTINUOUS SOUND RESTING STATE 18 measurements 20 measurements STRUCTURE

right left right left IC 12 10 5 6

LLN 10 9 6 2 SOC 10 8 2 4 CN 9 11 5 3

Table 1. Numbers of activated structures of brainstem.

pixel coordinates

pixel coordinates

Correlation map

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-0.04

-0.02

0

0.02

0.04

0.06

0.08MR : continous

Time [sec]

Signal change

ACRACLICR

Fig. 2. a) Correlation map for continuous sound: slice thickness 1.7mm, TR and white matter

as regressors. b) Time course from auditory cortex and brainstem (IC).

The shape of the curves from auditory cortex demonstrate similar behavior to curve of brainstem. Although signal changes from brainstem have lower amplitude than those from auditory cortex.

Presented fMRI acquisition technique allows to visualizing the auditory cortex, inferior colliculi, lateral lemniscus nuclei, superior olivary complex and cochlear nucleus. Some of this structures were also obtained during resting state measurements. Conclusion After application continuous stimulus (continuous sound) it was possible to detect cortical and subcortical structures as well as with using conventional block design paradigms. The difficulty in obtaining fMRI images was concealed in cardiac related pulsatile motion of brainstem. Others artifacts were partly eliminated by implementation signal changes from white matter as additional regressor.

a) b)

57

SOLID STATE NMR STUDIES OF COORDINATION AND ORGANOMETALLIC COMPLEXES OF NICKEL AND RUTHENIUM

Piotr Paluch and Marek J. Potrzebowski *

Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies,

Sienkiewicza 112, 90-363 Łódź, Poland.

Ni(II) with bis(acetylacetone)ethylenediamine ligand forms complexes which crystallizes as semi-hydrate (2) with C2/c space group in monoclinic system and anhydrous form (1) with Pna2(1) space group in orthorhombic system. 13C and 15N CP/MAS experiments were employed for structural characterization of both forms and searching of process of reversible water exchange in the crystal lattice. The applicability of ONIOM theoretical calculation in structural analysis of Organometallic Complexes (OMC) is discussed.

1) 2)

Ruthenium forms many useful organometallic complexes. In our study we concern on metathesis second generation Hoveyda-Grubbs catalyst (3) and their siloxyl derivatives (4). 13C CP/MAS ,1H-13C FSLG HETCOR, 1H-1H Back-to-Back (BABA), 2D CRAMPS will be presented. Problem with detection of carbon atom connected to ruthenium due to extremely large 13C CSA will be discussed.

Ru

OCH3

CH3

Cl

Cl

N

C(II)

NMesMes

Ru

OCH3

CH3

R3SiO

R3SiO

N

C(II)

NMesMes

3) 4)

58

1H, 13C, 15N NMR COORDINATION SHIFTS IN CATIONIC Fe(II), Ru(II), Os(II)

WITH 2,2’-BIPYRIDINE AND 1,10-PHENANTHROLINE

Leszek Pazderski,1 Tomasz Pawlak1, Jerzy Sitkowski,2,3 Lech Kozerski2,3, and Edward Szłyk1

1 Faculty of Chemistry, Nicholas Copernicus University, Toruń, Poland; 2 National Drug

Institute, Warsaw, Poland; 3 Institute of Organic Chemistry, Polish Academy of Sciences,

Warsaw, Poland

1H, 13C and 15N NMR studies of iron(II), ruthenium(II) and osmium(II) tris-chelated

cationic complexes with 2,2’-bipyridine and 1,10-phenanthroline of the general formula

[M(LL) 3]2+ (M = Fe, Ru, Os; LL = bpy, phen) were performed. Inconsistent literature 1H

signals assignments were corrected. Significant shielding of nitrogen-adjacent protons

(H(6) in bpy, H(2) in phen) and metal-bonded nitrogens was observed, being enhanced in

the series Ru(II) → Os(II) → Fe(II) for 1H, Fe(II) → Ru(II) → Os(II) for 15N, and bpy →

phen for both nuclei. The carbons are deshielded, this effect increasing in the order Ru(II)

→ Os(II) → Fe(II).

59

SEEKING A MOLECULAR SWITCH - STUDY ON 7-HYDROXY -4-METHYLQUINOLINE-8-CARBALDEHYDE IN SOLUTION

Mariusz Pietrzaka, Volha Vetokhinaa, Jacek Nowackib, Jerzy Herbicha,

and Andrzej L. Sobolewskic

a) Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland; b) Department of Chemistry, Warsaw University, Warsaw, Poland;

c) Institute of Physics, Polish Academy of Sciences, Warsaw, Poland Recently 7-hydroxy-4-methylquinoline-8-carbaldehyde (HMQC) was proposed as a candidate for an optically driven molecular switch [1]. IR and UV/VIS spectra of the isolated molecule in a low-temperature matrix as well as theoretical calculations indicate that this compound exhibits two tautomeric forms separated by a high barrier, which can be overcome by a photoprocess induced by UV radiation [1]. In this study the behaviour of the HMQC in solution is examined. Our aim is twofold. Firstly, we would like to confirm (or deny) the existance of the isomers proposed previously. Secondly, the characteristic of the system in solution is under study. UV and 1H NMR spectra in various solvents and at different temperatures were recorded and analysed. The N-H isomer was identified in the solution. Our results show, however, that both isomers are in thermal equilibrium depending on the temperature and the solvent. Mechanism of this phenomenon, which is in apparent contradiction to the low-temperature matrix results, is discussed.

N

CH3

O

HOH

N

CH3

O

OHH

in solution

[1] L. Lapinski, M. J. Nowak, J. Nowacki, M. F. Rode, A. L. Sobolewski, A Bistable Molecular Switch Driven by Photoinduced Hydrogen-Atom Transfer, ChemPhysChem 2009, 10, 2290-2295. Scientific work financed from the science funds for the years 2008-2011 as a research project.

60

SPIN-LATTICE RELAXATION IN DOPA-MELANIN AND MELANIN ISOLATED FROM Sepia officinalis – COMPARATIVE EPR STUDIES

Barbara Pilawa1, Magdalena Zdybel1, Daria Czyżyk1, Ewa Chodurek2, and Sławomir

Wilczyński1

1,2Medical University of Silesia in Katowice, School of Pharmacy and Division of Laboratory

Medicine, 1Department of Biophysics, Sosnowiec, Poland; 2Department of Biopharmacy, Sosnowiec, Poland

Spin-lattice relaxation in model and natural eumelanins was studied by an X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy. Synthetic DOPA-melanin as the model eumelanin and eumelanin isolated from Sepia officinalis were tested. The melanin samples were obtained from Sigma Firm. Chemical structure of eumelanin is presented in Figure 1 [1]. Melanin biopolymer from Sepia officinalis is used in cosmetics [2] and the knowledge about its paramagnetic centers system is very important to cosmetology, dermatology, and esthetical medicine. The aim of this work is to compare magnetic interactions in DOPA-melanin and melanin isolated from Sepia officinalis.

NH

NH

(COOH)

OH

OH

NH

(COOH)

O

O

(COOH)HOOC

O

Fig. 1. Chemical structure of eumelanin [1].

EPR measurements were done by the use of electron paramagnetic resonance spectrometer of RADIOPAN Firm (Poznań). The first-derivative EPR spectra were recorded with microwave power in the range of 0.7-70 mW. Changes of amplitudes (A) and integral intensities (I) of EPR spectra with increasing of microwave power were evaluated. Amplitudes and integral intensities of EPR spectra of the studied melanins increase with increasing of microwave power, and after reaching the maximum they decrease for the higher microwave powers. Spin-lattice relaxation time increases with decreasing of microwave power of saturation of EPR spectra of the sample [3]. It was shown the slow spin-lattice relaxation processes exist in the both examined eumelanins – DOPA-melanin and melanin from Sepia officinalis. References: [1] Wakamatsu K, Ito S. Advanced chemical methods in melanin determination. Pigment Cell Res 2002; 15: 174-183.

61

[2] Gibka J. Wykorzystanie melaniny i procesu melanogenezy w kosmetyce. Polish Journal of Cosmetology 2000; 3: 164-176. [3] Stankowski J, Hilczer W. Wstęp do spektroskopii rezonansów magnetycznych. Warszawa: PWN; 2005. Acknowledgements: This study was supported by Medical University of Silesia in Katowice (Grant no: KNW 1-142/09).

62

SOLID-STATE CHARACTERIZATION OF S(+)CLOPIDOGREL HYDROGENSULPHATE PHARMACEUTICALS AND THEIR

POLYMORPHS

Edyta Pindelska, Andrzej Mazurek, and Wacław Kołodziejski

Department of Inorganic and Analytical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland

The importance of polymorphism and solvate formation in the crystallization of organic compounds has an increasing importance in the pharmaceutical industry because those substances may have chemically identical, but very different physical and biological properties. Polymorphism is defined as the ability of a substance to exist as two or more crystalline phases that have different arrangements and/or conformations of the molecules in the crystal lattice.1 The “polymorphs of a solvate” means that two solvates of the same compound with identical solvent molecules and the same stoichiometry can exist in different crystal structures.2 S(+)clopidogrel hydrogensulphate - CL (Figure 1) is a pharmaceutical compound with a novel mechanism of action in the prevention of atherosclerotic events. CL is an example of polymorph of a solvate. At least six polymorphic forms are known for CL, but only two of them (form I and II) are used in pharmaceutical formulations.3

Figure 1. Structural formulae of S(+)clopidogrel hydrogensulphate (CL)

The solid-state studies of the two bioactive polymorphs (I and II) and commercial

available preparations: Plavix®, Areplex® and Zylt® were carried out using 13C and 15N CP/MAS NMR spectroscopy and powder XRD analysis, additionally supported by ab initio calculations. The assignment of the solid-state NMR spectra was done by comparison of chemical shifts in solid and liquid phases as well as by analysis of CP kinetics and dipolar-dephased spectra.4

Comparison of the 13C CP/MAS spectra of pure polymorphic forms I and II to those from tablets proved that Plavix® contains polymorph II as an active substance, while Areplex® and Zylt® contains polymorph I. Our study proves that 13C CP/MAS technique is sensitive enough for determination of polymorphic content in pharmaceutical tablets.

1 J. Bernstein, In Oxford University Press, Polymorphism in Molecular Crystals, Clarendon Press, Oxford 2002; 2 J. Bernstein, Cryst. Growth. Des. 1661, 5, 2005 ; 3 A. Bousquet, B. Castro, S. Germain, Polymorphic Form of Clopidogrel Hydrogen Sulphate. US Patent no. 6504030, 2003; 4 W. Kołodziejski, J. Klinowski, J. Chem. Rev. 102, 613, 2002;

63

CONSTRUCTION OF HIGH FREQUENCY COILS FOR MRI AT 0.088 T

Bartosz Proniewski1, Henryk Figiel1, and Tadeusz Pałasz2

1 University of Science and Technology, Faculty of Physics and Applied Computer

Science, Kraków, POLAND 2 M. Smoluchowski Institute of Physics, Jagiellonian University

Kraków, POLAND

While most of the research in magnetic resonance imaging is focused on the development and applications of high field systems, there are certain areas of diagnostic medicine where imaging at low magnetic field strengths yields comparable results. Utilization of such systems requires specially designed dedicated rf coils in order to overcome the technical constrains resulting from working at low magnetic fields. The main purpose of this work was to determine whether it was possible to image an elbow at 0.088T using different types of coils not evaluated in this particular system yet. For the purpose of this project a total of four different coils have been designed, constructed and tested, namely two homogeneous resonators: the solenoid and saddle shape coils, and two heterogeneous resonators: the basic planar surface coil and half saddle surface coil. The design process consisted of determining the desired coil dimensions using available anthropometric data, as well as coil inductance and magnetic field simulations. A series capacitive network has been used to match the designed coils to the spectrometer at the desired frequency and Q factors have been evaluated using a spectrum analyzer. Magnetic field induction generated by the coils has been measured and compared with theoretical expectations and the theoretical field homogeneity has been assessed using the field histogram method. Images of six phantoms have been acquired using the constructed coils, which allowed signal to noise ratio measurements and field profiles to be determined and compared with field simulations. After the tests with phantoms, images of an elbow have been successfully obtained using all of the constructed coils. The solenoid, saddle and planar surface coils provided good overall image quality. Additionally the ability to focus the imaging plane at the desired depth with surface coils has been demonstrated. In was shown that imaging at very low fields can be successfully performed with other than the solenoid coil configuration.

64

EPR STUDIES OF PARAMAGNETIC PROPERTIES OF THERMALLY TREATED SISOMICIN AND VERAPAMIL

Paweł Ramos, Barbara Pilawa, and Piotr Pepliński

Medical University of Silesia in Katowice, School of Pharmacy and Laboratory Medicine,

Department of Biophysics, Sosnowiec, Poland,

Properties of paramagnetic centers in thermally treated sisomicin and verapamil were compared. Sisomicin is an aminoglycoside antibiotic [1-2]. Verapamil is an L-type calcium channel blocker [1-2]. Chemical structure of the studied drugs is presented in Figure 1 [1]. These two drugs were heated in dry air at the following temperatures: 160oC, 170oC, and 180 oC. Times of heating of the samples were: 120, 60, and 30 minutes, respectively. The aim of this work is to compare effect of thermal sterilization on paramagnetic centers formation in sisomicin and verapamil. Electron paramagnetic resonance (EPR) spectroscopy was the experimental method.

OO

NH2

O

NH2

NH2

NH2

O

OH

NH

OH

OH

N

N

OO

O

O

Fig. 1. Chemical structure of sisomicin (a) and verapamil (b) [1].

EPR spectra were measured for all the heated samples. Amplitude (A), integral intensity (I), and linewidth (∆Bpp) of the EPR spectra were analysed. g-Factors were calculated. Spin-spin and spin-lattice interactions in the heated drugs were characterized. Concentrations of paramagnetic centers in sisomicin and verapamil were determined. The obtained results are useful to optymalization of thermal sterilization process of these drugs. References: [1] A. Zejc and M. Gorczyca (Red.), Chemia leków, Wydawnictwo Lekarskie PZWL, Warszawa, 2002 [2] W. Janiec and J. Krupińska (Red.), Farmakodynamika. Podręcznik dla studentów farmacji. Wydawnictwo Lekarskie PZWL, Warszawa, 2002 Acknowledgements: This study was supported by Medical University of Silesia in Katowice (Grant no: KNW 1-142/09).

65

THE INFLUENCE OF TRACES OF WATER ON THE CHEMICAL SHIFTS OF SQULAENE IN BENZENE SOLUTION

Michał Rażew, Ewa Kaczorowska, Ewa Kula-Świeżewska, Ewa Ciepichał, and Jacek Wójcik

Instytut Biochemii i Biofizyki PAN, Warszawa, Poland Squalene is a linear polymer composed of six isoprene units (Fig. 1). Squalene commonly occures in nature. It is produced by all higher organisms and plays an important biological role as a precursor in the synthesis of all steroids (i.a. cholesterol), as a component of human sebum with antibacterial and antifungal properties or a compound stimulating the immune system and increasing the response to a vaccine (immunologic adjuvant). At present, the Novartis company use squalene as a component of MF59-adjuvanted influenza vaccine [1], its usefulness in the malaria vaccine is also researched. Squalene has been found effective in inhibiting the processes of cancer of the skin and lungs of rodents [2]. Squalene is also used in cosmetics as an antioxidant and a lubricant.

Figure 1. The scheme of squalene.

In the NMR spectra of squalene the 13C signals are grouped in several ranges: signals of methyl groups at 10-30 ppm, signals of methylene gropups at 25-50 ppm and signals of methine groups at 120-130 ppm. Because of the similar chemical character and surroundings of most of carbon nuclei within a given functional group, the signals arising from different nuclei overlap impeding interperation of 13C NMR spectrum. In this work the influence of traces of water on the chemical shifts of carbon and proton is presented. The results are shown at the example of 1H - 13C correlation spetra. The HSQC spectrum of squalene sample at a concentration 1µM in C6D6 was recorded an a Varian Inova 400 MHz spectrometer at 25°C. 1H and 13C chemical shifts were compared with those obtained under the same conditions, but with water addition. The calculations of the shielding constants of carbon and hydrogen atoms in the presence of benzene and mixture of benzene and water were also performed. The calculations of shielding tensors and geometry optimization were carried out using Gaussian 03 program with HF method.

References: 1. MF59 Adjuvant Fact Sheet, Novartis, June 2009. 2. Desai, K.N.; Wei, H.; Lamartiniere, C.A. The preventive and therapeutic potential of the squalene-

containing compound, Roidex, on tumor promotion and regression. Cancer Lett. 1996, 19, 93-96, Molecules 2009, 14 552.

66

APPLICATION OF MRI FOR SPINAL CORD MONITORING IN ANIMAL MODEL OF PRESSURE IMPACT INJURY

Paulina Rosicka1, Katarzyna Majcher1, Wiesław Marcol2, Wojciech Ślusarczyk2, Tomasz Banasik1, Joanna Lewin-Kowalik2, and Władysław P. Węglarz1

1 H. Niewodniczański Institute of Nuclear Physics PAN, Kraków, Poland

2 Silesian Medical University, Katowice, Poland

After the spinal cord incident, the acute impact causes the primary injury and long lasting processes of secondary damage which occur weeks later. Time dependent changes of the water properties in the injured neural tissue may help to understand these processes, as well as monitor effects of the of neuroprotective drugs treatment.

The experiment was performed on adult male rats. The pressure impactor was used to impair neural tissue in lower cervical cord. The animals were divide to two groups with different neuroprotective drug applied to each. As neuprotective substances nicotine and N-acetyl-cysteine were used. At different time after injury (from 2 weeks to 2 months) MRI measurement were done on 4.7 T system using SE sequence . The data from 15 axial slices chosen between 10 Th and 12 Th spine level was aqcuired. The T2- and DWI – weighted images were obtained with diffusion gradients applied parallel and perpendicular to the spinal cord.

MR images free from any motion artifacts were obtained from spinal cord in vivo. Maps of diffusion coefficients (longitudinal and transverse diffusion) were calculated for axial slices. Comprehensive analysis of diffusion and T2- weighted data from ten ROI’s in gray and white matter gives quantitative description of injury in terms of spatial and temporal changes of the diffusion parameters.

67

INTERACTION OF SELECTED BICYCLIC MONOTERPENES WITH αααα-CYCLODEXTRIN.

THERMODYNAMIC CHARACTERISTIC OF COMPLEXATION

Katarzyna Ruszczyńska-Bartnik1, Michał Nowakowski1, Helena Dodziuk2,

and Andrzej Ejchart1

1 Laboratory of Biological NMR, Institute of Biochemistry and Biophysics, Polish Academy of

Sciences, Warszawa, Poland; 2 Institute of Physical Chemistry, Polish Academy of Sciences, Warszawa, Poland

Chiral recognition by cyclodextrins, CDs, is of considerable importance in the aspect of enantiospecific action of many drugs. The mechanism of chiral recognition is not yet fully understood and, therefore, determination and comparison of thermodynamic data for variety of diastereomeric complexes is necessary. NMR spectroscopy is the method that is well suited for studying stoichiometry, association constants and geometry of inclusion complexes. Usually association constants of weak supramolecular complexes are determined under assumption of chemical exchange between free and complexed states which is fast on the chemical shift timescale. In such a case the observed NMR parameter value is the mole fraction weighted average of the parameter values in the free and complexed molecule. However, many of bicyclic monoterpens and α-CD form diastereomeric complexes with stoichiometry 1 : 2 which do not fulfill this assumption. Moderately fast chemical exchange in these complexes results in the broadening of resonances and precludes direct read-out of their frequencies from NMR spectra. Unjustified assumption of the mole fraction weighted average may, under certain circumstances, lead to significant underestimation of association constant and, in consequence, to non-negligible errors in Gibbs free energy under determination. Simultaneous fit of theoretical exchange broadened lineshapes to all NMR spectra measured in a titration experiment is the only correct procedure which allows one to determine not only non-biased association constant Ka but also exchange rate k [1, 2]. Despite the more complicated data analysis the exchange broadened spectra deliver information on the activation parameters of complexation processes which is unavailable in the fast exchange limit.

Our study of diastereomeric complexes formed between bicyclic monoterpens and α-CD includes camphor [2], borneol [3] and isoborneol. Their structures are shown in Fig. 1. Figure 1

68

Methodology of the determination of association constants and exchange rates for the studied complexes from the exchange broadened 1H NMR spectra has been described elsewhere [2]. Ka and k parameters can be used to calculate Gibbs free energies ∆G0 and ∆G# (see Fig. 2) characterizing complex formation according to formulae: )ln(0 aKRTG −=∆

)ln(#

Tk

hkRTG

B

−=∆

Figure 2 The final results are given in Table 1 Table 1.

1R4R enantiomer 1S4S enantiomer Bicyclic monoterpene ∆G0 [kJ/M] ∆G# [kJ/M] ∆G0 [kJ/M] ∆G# [kJ/M]

∆∆G0 [kJ/M] (1S4S-1R4R)

camphor –53.6±0.2 58.82±0.03 –51.4±0.1 59.38±0.02 2.2±0.2 borneol –51.9±0.1 57.19±0.03 –50.9±0.1 57.39±0.03 1.0±0.1 isoborneol –53.4±0.1 56.37±0.06 –53.2±0.1 56.88±0.06 0.2±0.1 Absolute configuration of C1 and C4 chiral centers determines relative stability of studied diastereomeric complexes. Enantiomers 1R4R form more stable complexes with α-CD than the 1S4S ones. Absolute configuration of an additional chiral carbon C2 does not influence this regularity. ∆∆G0 may be regarded as a measure of chiral recognition. It is the largest for camphor and the smallest for isoborneol. On the other hand, Gibbs free energies of activation, ∆G#, are larger for the 1S4S containing complexes. In the diastereomeric pairs larger activation energy corresponds to less stable complex. References: [1] J. Feeney, J.G. Batchelor, J.P. Albrand, G.C.K. Roberts (1979) The effects of intermediate

exchange processes on the estimation of equilibrium constants by NMR. J. Magn. Reson., 33, 519-529.

[2] P. Bernatowicz, M. Nowakowski, H. Dodziuk, A. Ejchart (2006) Determination of association constants at moderately fast chemical exchange. Complexation of camphor enantiomers by α-cyclodextrin. J. Magn. Reson., 181, 304-309.

[3] K. Ruszczyńska-Bartnik, M. Nowakowski, H. Dodziuk, A. Ejchart (2008) NMR titration at intermediate exchange rate conditions: borneol – α-cyclodextrin system. NMR in Chemistry, Physics and Biological Sciences. 24-26.09.2008, Warszawa. Abstract Book, P-22.

69

SODIUM MR IMAGING

Lothar R. Schad

Lehrstuhl für Computerunterstützte Klinische Medizin, Medizinische Fakultät Mannheim, Universität Heidelberg, Mannheim, Germany

Sodium (23Na) ions play an important role in cellular homeostasis and cell viability. In

healthy tissue, the extracellular sodium concentration ([Na+]ex = 145 mM) is about ten times higher than the intracellular concentration ([Na+]in = 10-15 mM) [1]. Using sodium MRI, volume- and relaxation-weighted signal of these compartments can be measured. Thus, sodium MRI is a promising diagnostic tool, since pathological processes can alter this ion gradient. A density adapted 3D radial projection reconstruction pulse sequence (DA-3DPR) is presented [2] that provides a more efficient k-space sampling than conventional 3D projection reconstruction sequences (3DPR). The gradients of the DA-3DPR sequence are designed such that the averaged sampling density in each spherical shell of k-space is constant. Due to hardware restrictions, an inner sphere of k-space is sampled without density adaption. Benefits for low SNR applications are demonstrated with the example of sodium imaging. In simulations of the point-spread function (PSF), the SNR is increased by the factor 1.66 for the DA-3DPR sequence. Using analytical and experimental phantoms, it is shown that the DA-3DPR sequence allows higher resolutions and is more robust in the presence of field inhomogeneities. High quality in vivo images of human brain, muscle and kidney are acquired at 3.0 and 7.0 Tesla. For equivalent scan times, up to a factor of 1.80 higher SNR is observed and anatomical details are better resolved using DA-3DPR. References: 1. Hilal SK, Ra JB, Oh CH, Mun IK, Einstein SG, Roschmann P. Sodium Imaging. In: Stark

DD, Bradly WG, editors. Magnetic resonance imaging. St. Louis: C.V. Mosby; 1988. p. 715-729.

2. Nagel AM, Laun FB, Weber MA, Matthies C, Semmler W, Schad LR. Sodium-MRI using a density adapted 3D radial acquisition technique. Magn. Reson. Med. 2009, in print.

70

THE CHANGES OF PHOSPHOLIPIDS IN LIPID RAFTS AT PATIENTS WITH MYELODYSPLASTIC SYNDROME (MDS)

Joanna Schiller1, Małgorzata Kuliszkiewicz-Janus1, Izabela Dereń-Wagemann1, and Stanisław Baczyński2

1 Department of Hematology and Transplantology, Wrocław Medical University, Poland, 2 Faculty of Chemistry, University of Wrocław, Poland

INTRODUCTION The myelodysplastic syndromes (MDS) are a heteregenous group of clonal hematopoietic stem cell disorder. They are characterized by two main features: an impaired maturation (dysmyelopoeiesis) and peripheral blood cytopenias resulting from ineffective hematopoiesis. Recent investigation has shown that impaired lipid rafts occur in neutrophils of patients with MDS. The lipid rafts are involved in signal transduction, endocytosis, and cholesterol transport in cells. They are specialized subdomains of the plasma membrane enriched in cholesterol, sphingomyelin, gangliosides and certain proteins. Phospholipids, which built the lipid rafts, contained saturated fatty acyl chains which tend to be more tightly packed. Together with high concentration of cholesterol it results in high order and less fluidity then other part of the membrane. Lipid rafts can be extracted from cell membranes as detergent-resistant memebranes (DRMs), when we use for example 1% Triton-X 100. AIMS To continue our previous studies on the application of 31P MRS in vitro to the analysis of phospholipids changes in erythrocytes from patients with MDS, we decided to evaluate this method to examine concentration of phospholipids in lipid rafts and disturbances in rafts flotation in a 10%- 40% linear sucrose density gradient. MATERIALS AND METHODS The investigation was carried out on erythrocytes from patients suffering from MDS (6 patients) and healthy volunteers (6 persons). The DRMs were isolated from erythrocytes with modified R. Prohaska procedure [3]. Fractions (500µL) were collected from the top. Phospholipids

were extracted with a modified Folch method. The spectra 31P were obtained on AMX 500 Bruker (7.05T) spectrometer. Calculation of phospholipid concentration was based on peak integral

intensities in the 31P NMR spectra for the individual compounds and methylenediphosphonic acid (MDPA). RESULTS

The DRMs from healthy volunteers were always isolated in 2nd fraction. However in patients

with MDS they were isolated in 3rd, 4th, 5th and 6th fraction. It may depends on the staging of the disease (IPSS). The lower fraction in which the DRMs occur may be dependent on advance of

disease. We observed that, the 31P spectra of phospholipids extracts consisted of 3 resonant peaks. Two due to phospholipids: PC and SM and another one – due to external reference substance

71

MDPA. Our preliminary data showed that in patients with MDS there is a tendency of decrease of SM peak and increase of PC peak in comparison with healthy volunteers. CONCLUSION Changes observed in phosphholipids concentrations in lipid rafts from eythrocytes in patients with MDS may result from dyserythropoeisis, which can be observed in this disease. Decrease in sphingomyelin concentration in this patients may disturb structure of lipid rafts plasma membrane. It may result that abnormal erythrocytes are being removed or destroyed which can explain anemia by hypercellular bone marrow.

72

RELAXATION PROCESSES OF SELECTED BIOMOLECULES

Aleksandra Skowronska and Danuta Kruk

Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Krakow, Poland

In this work relaxation properties of water solutions (H2O and D2O) of selected

biomolecules are discussed. Due to the nature of the problem the temperature window of the

relaxation studies is rather narrow. Therefore to observe reorientational dynamics of

biomolecules viscosity of the solutions has been adjusted by adding different amounts of

glycerol. Slow motion of large molecules is investigated at low magnetic fields in order to

keep the condition that the Zeeman splitting (in angular frequency units) is comparable to a

correlation time reflecting the time-scale of the molecular dynamics. The issue of anisotropic

motion of large biomolecules in viscous solutions is discussed.

This work has been financed by Polish Ministry of Science and Education, grant No N N202 105936

73

IMPLEMENTATION OF RETROSPECTIVE GATING IN APPLICATION TO MR ASSESSMENT

OF CARDIAC FUNCTION IN MICE

Tomasz Skórka1, Sylwia Heinze-Paluchowska1, Urszula Tyrankiewicz1, and Magdalena Jabłońska 1,2

1-Zakład Tomografii MR, Instytut Fizyki Jądrowej PAN, Kraków; 2Katedra Fizyki Medycznej i Biofizyki, Wydz. Fizyki i Informatyki Stosowanej AGH, Kraków

MRI of the heart is sensitive to cardiac and respiratory motions, what may cause several artifacts in acquired images. In order to achieve images with proper mapping of the heart ventricle volumes data acquisition is synchronized with the heart motion using ECG or intrinsic NMR signal (self-gating method). These measurements must be synchronized with ECG signal to achieve. It’s very important, especially in cine imaging, where many images per cardiac cycle are achieved. As commonly used, prospective gating has many advantages, but isn’t possible to cover the entire cardiac cycle - there is a lapse of time at the end of the diastole. This problem can be eliminated by the use of the retrospective gating method, where MRI data acquisition is continuous with a simultaneous ECG recording to reorganize the data during image reconstruction (data are collected regardless of the cardiac cycle).

In this work we present the implementation of the retrospective triggering method on the MARAN DRX console. Detection of the R-wave is performed with the use of dedicated device (SA Instruments Ltd, USA). Written software (IDL, ITT USA) enable for estimation of the mean heart rate, than the consecutive R-waves are detected and assigned them to the proper position on the time axis. The information about cardiac motion during imaging is constantly recorded. After completion of data acquisition, this information is used for interpolation and sorting of the data according to the corresponding phases of the cardiac cycle. A number of images depicting different stages of the mouse heart motion were collected and reconstructed using dedicated software.

Implemented method of reconstruction gives ability for obtaining images from the entire cardiac cycle for easy changing number of reconstructed images, and potentially can be more accurate according to the possibility of the R-wave timing post-processing. However, ECG-based reconstruction is in general less useful in situations, when heart rate isn’t constant (arrhythmia) or ECG signal is so disturbed that we are unable to detect it in a proper way (transgenic mice). In these cases the best solution seems to be a method called “self-triggering”.

Acknowledgments: This work was supported by PMSHE, grant NN 518419733 (2007-2010)

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INFLUENCE OF PARAMAGNETIC IONS PRESENCE ON RELAXATION IN BLOOD SERUM

Lech Skórski, Mateusz Synowiecki, and Barbara Blicharska

Jagiellonian University, Department of Physics, Krakow, Poland

The presence of paramagnetic ions like Cu, Mn, and Fe influences on the relaxation

times in biological systems. This fact can be used to estimate concentration of free paramagnetic ions in blood serum. As an exemplary application, we present the results of the proton spin–lattice relaxation times measurements in blood serum before and after chelation, which may be used as an alternative method for monitoring the presence of free copper ions This method may be used in the diagnosis of diseases like leukaemia, liver diseases, and particularly Wilson’s disease. The advantage is that in contrast to conventional methods, like spectrophotometry, which records the total number of both bound and free ions, the proton relaxation technique is sensitive solely to free paramagnetic ions dissolved in blood serum.

75

THE ITERATIVE APPROACH TO PROCESSING OF RANDOMLY SAMPLED 3D NMR SPECTRA

Jan Stanek, and Wiktor Koźmiński

Faculty of Chemistry, University of Warsaw,

Warsaw, Poland

Multidimensional (nD) NMR has become the routine method in research on the structure and dynamics of biomolecules. Classical approach to nD-signal sampling and processing that employs Discrete Fourier Transform (nD DFT), has been gradually replaced by alternative methods, which use more or less random sampling schemes of evolution time domain and therefore are not limited by the Nyquist theorem [1]. These new methods are primarily applicable to sensitive (i.e. featuring high S/N) nD experiments. Their main advantage is the enhancement of spectral resolution achieved in the reasonable experimental time. However, irrespective of the particular method, obtained spectra contain artifacts [2]. They can be substantially suppressed by (a) optimization of the sampling schedule [3] and/or (b) application of a specialized method of processing. Recently discovered Multidimensional Fourier Transform seems a very elegant, linear and general method, however, it is not suitable for spectra of high dynamic range. In this work the fully automatic iterative cleaning procedure is applied in order to remove artifacts. It employs a linear and exactly reversible transformation (SVD assisted FT) and original signal detection algorithm. The benefits of the new approach have been proved experimentally on 3D 15N and 13C-edited NOESY-HSQC spectra of human ubiquitin, which have been obtained using ca 5 % of samples. References: [1] K. Kazimierczuk, A. Zawadzka, W. Koźmiński, I. Zhukov, Random sampling of evolution time space and Fourier transform processing, J. Biomol. NMR 36 (2006) 157-168; [2] K. Kazimierczuk, A. Zawadzka, W. Koźmiński, I. Zhukov, Lineshapes and artifacts in Multidimensional Fourier Transform of arbitrary sampled NMR data sets, J. Magn. Reson. 188 (2007) 344 – 356; [3] K. Kazimierczuk, A. Zawadzka, W. Koźmiński, Optimization of random time domain sampling in multidimensional NMR, J. Magn. Reson. 192 (2008) 123-130.

76

STRUCTURE AND STEREOCHEMISTRY OF NTBC

AND ITS METABOLITES - A COMBINED NMR/DFT STUDY

Przemysław Szczeciński, Anna Dziadecka and Adam Gryff-Keller

Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warszawa, Poland

NTBC, i.e. 2-[2-nitro-4-(trifluoromethyl) benzoyl]cyclohexane-1, 3-dione (Fig. 1), is a lifesaving drug against an inherited metabolic disease - tyrosinemia type I. It has been found that this drug is metabolized in organisms of patients and excreted in urine as 5-hydroxy-NTBC and 6-hydroxy-NTBC (Fig. 1). After separation and chemical identification the molecular structures of the NTBC metabolites have been investigated in detail using 1H, 13C and 19F NMR spectroscopy and quantum chemical calculations. The investigated compounds belong to the class of β,β-triketones that in solutions can undergo various tautomeric and conformational transformations. The structures of the most abundant forms of the investigated compounds in CDCl3 solutions have been established.

Figure 1. NTBC and its metabolites. The molecular geometry optimizations were done by DFT (PBE1PBE/6-31+G*) method, whereas the theoretical values of NMR parameters were calculated by GIAO-DFT method using PBE1PBE/6-311++G(2d,p) level of the theory. The impact of the solvent was taken into account by PCM method at both calculation stages.

O

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77

MOLECULAR DYNAMIC AND STRUCTURE OF PHOSPHATYDYLOCHOLINE (DMPC)/CATIONIC GEMINI

SURFACTANT (GEM-IK1) SYSTEM

Kamil Szpotkowski, Aleksandra Wypych, Kosma Szutkowski, ad Maciej Kozak, Stefan Jurga

Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Poznań, Poland

Phospholipids are structural basis of biological membranes, with structures strongly dependent on temperature and additions, such as cholesterol and surfactants [1]. Gemini surfactants are a new generation amphiphilic molecules containing two monomer surfactant molecules linked by a hydrophilic spacer. This group of surfactants have special properties and potential applications in many areas [2]. Gemini surfactant molecule intercalates between zwiterionic phospolipid molecules so that the total surface charge is altered which manifests in change of the structure of the bilayer [3]. Aqueous suspensions of 1,2-dimyrilstoyl-sn-glycero-3-phosphocholine (DMPC) with different concentration of the surfactant 1,1’-(1,4-butane) bis 3-decyloxymethylimidazolium chloride (GEM-IK1) have been investigated by Small Angle X-ray Scattering (SAXS), Pulsed Field Gradient NMR (PGSE NMR), Broadband Dielectric Spectroscopy and Fourier Transform Infrared Spectroscopy. The broad distribution of diffusion coefficients was found for pure DMPC, while addition of gemini surfactant manifested in the significant narrowing of the distribution of diffusion coefficients as the result of a change in the size distribution of aggregates (1A). This conclusion is supported by SAXS diffraction patterns (1B). Dielectric spectroscopy in frequency range from 106 Hz to 109 Hz was used to study polar part of DMPC molecule(2A). The observed step-like change in the dependence of dielectric constant vs. temperature is explained by a transition from gel to liquid crystalline phase (2B). This is further confirmed by temperature dependence of stretching bands obtained by FTIR for phosphate and carbonyl groups. Two characteristic bands from IR spectra, corresponding to symmetric and antisymmetric vibrations of methyl groups, were observed to detect changes in the non-polar part of phospholipid bilayers. Our results show that the transition from lamellar to bicellar phase is indeed induced by the addition of the surfactant. Furthermore the temperatures of observed main- and pre-transitions are decreasing with an increase of the surfactant concentration.

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Figure 1. DMPC and DMPC/GEM-IK1 (0.1%, 0.5%, 1%, 5%) systems at 293K. Log-log plot of a spin-echo amplitudes as obtained from PGSE NMR (1A). SAXS diffraction patterns (2A).

Figure 2. Dielectric permittivity vs. frequency for 10% DMPC suspension (2A). Dielectric

spectra of ε’ vs. temperature for DMPC/GEM-IK1/H2O systems, with different amounts of GEM-IK1 measured at 1.5 MHz.

References: [1] J. Katsaras, T. Gutberlet (Eds.), Springer- Verlag, Berlin – Heidelberg, 2001. [2] Mingqi Ao, Guiying Xu, Yanyan Zhu, Yan Bai, Journal of Colloid and Interface Science 326 (2008) 490–495. [3] D. Uhrikowa, G. Rapp, P. Balgavy. Bioelectrochemistry 58 (2002) 87-95. Acknowledgements: This work is supported by 6th Framework Programme under SoftComp Grant No 502235-2 and research grant No N202 248935 (Poland). A. Wypych expresses her appreciation to the Ministry of Science and Higher Education in Poland for a Postdoctoral Fellowship (POL-POSTDOC III).

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79

MOLECULAR DYNAMICS IN PROTON CONDUCTING GEL/H3PO4 ELECTROLYTES STUDIED BY NMR

L. Szutkowska, M. Dobies, K. Szutkowski, Stefan Jurga, G. śukowska*, and W. Wieczorek*,

Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University,

Poznań, Poland; *Faculty of Chemistry, Warsaw University of Technology, Warszawa, Poland

Migration mechanisms and dynamics of protons are crucial for understanding the

phenomena of the proton conductivity in novel nonaqueous polymer systems. Recently the new nonaqueous electrolytes have been proposed [1, 2], which are doped with phosphoric acids. These systems can be used in polymer electrolyte type of fuel cells operating at low humidity conditions and high temperatures above 100oC. We have examined nonaqueous gel consisting of phosphoric acid H3PO4 (PA) dissolved with propylene carbonate (CP), entrapped within polymer matrix, formed from poly(methylmethacrylate) (PMMA). Two samples were under investigation with the same amount of PMMA (12% wt.) but with different content of organic solution of CP/H3PO4 (81/7 and 48/40 % wt.).

Both samples were investigated by NMR. Slow molecular dynamics was studied by means of T1 relaxation times as a function of Larmor frequency in the Fast–Field Cycling NMR experiment. A strong dispersion of relaxation rate 1/T1 = R1 (ω), especially at high-frequency range from 200 to 1 MHz, was observed for gel with higher H3PO4 content. For the gel with a lower concentration of H3PO4, the dispersion R1(ω) is becoming weaker. The relaxation behavior may be explained by different interactions (adsorption mechanism) of organic solution of CP/H3PO4 with a polymer matrix in both samples. Weak adsorption obviously leads to flat, however, strong adsorption - to steep spin-lattice relaxation dispersions [3, 4]. The translational diffusion was measured by Pulsed Field Gradient Stimulated Spin Echo (PGSTE) NMR. Diffusion coefficients turned out to be dependent on diffusion time ∆, giving an evidence for restricted diffusion. Furthermore diffusion coefficients for all contributing proton species were analyzed by using pseudo 2D displays of Diffusion Ordered Spectroscopy (DOSY). All results are discussed with respect to the ionic conductivity of studied gel electrolyte systems [5,6]

References [1] M. Schuster, T. Rager, A. Noda, K. D. Kreuer, J. Maier, Fuel Cells, (2005), 5, 355. [2] K. D. Kreuer, S. J. Paddison, E. Spohr, M. Schuster, Chem. Rev., (2004), 104, 4637. [3] S. Stapf, R. Kimmich, and R. O. Seitter, Magn. Reson. Imaging., (1996), 14, 841. [4] J.-C. Perrin, S. Lyonnard, A. Guillermo, and P. Levitz, J. Phys. Chem., (2006), 110, 5439. [5] K. R. Jeffrey, W. Wieczorek, D. Raducha, and J.R. Stevens, J. Chem. Phys., (1999), 110 (15), 7474. [6] K. R. Jeffrey, G. Z. śukowska, J. R. Stevens, J. Chem. Phys., (2003), 119 (4), 2422.

80

METABOLIC PROFILE OF CEREBROSPINAL FLUID OBTAINED FROM AMYOTHROPHIC LATERAL SCLEROSIS PATIENTS

Beata Toczylowska Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland;

Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

Introduction Aim of this study is to determine metabolic profiles of cerebrospinal fluid (CSF) obtained from the Amyotrophic Lateral Sclerosis (ALS) patients and to compare them against metabolic profile of samples obtained from control group. CSF surrounds the brain and fills the ventricles as well as the subarachnoid space. It is the secretion product of various central nervous system structures and its ultimate composition depends on exchange of metabolites with the blood and adjacent brain tissue. Since the composition of CSF is directly dependent upon metabolite production rates in the brain, analysis of the CSF metabolic profile can offer biochemical insights into central nervous system disorders.

Methods CSF used for our examination was collected by lumbar puncture from lumbar subarachnoid space. Two sample categories were collected: control, neurologically healthy, subjects (n=15) and ALS patients (n=18). Patients without neurological diseases, who underwent varices or inguinal hernia surgery under spinal anesthesia were qualified to the control group. CSF samples were centrifuged at room temperature at 15000 rpm for 5 minutes, and then frozen at -80°C until NMR analysis was performed. In the NMR experiments solvent suppression was used to minimise strong water signal. 512 scanes and 12 s pulse sequence repetition were used at 25 C. Small amount of D2O was added to each sample in order to provide lock signal. 1mM trimethylsilyl propionate (TSP) was used as a chemical shift reference. TSP signal was also used for normalisation of all spectra enabling quantitative statistical and discrimination analyses. Amounts of metabolites were expressed as relative intensities of their spectral peaks determined in comparison to the TSP peak, because absolute concentrations were difficult to determine using the analytical techniques applied here. Each NMR spectrum was reduced to smaller number of variables, using the target profiling method. Twenty eight signals from each spectrum were analyzed. Multivariate partial least square discriminant analysis (PLS-DA) was performed using the software package SIMCA-P(Version 12, Umetrics AB, Umeå, Sweden)(1).

Results For the multivariate analysis 28 signal of the following components identified in NMR spectra were selected: formate, histidine, phenylalanine, tyrosine, myo-inositol, unassigned signals at δ=4.03 and δ=1.08, glucose, glycine, scyllo-inositol, choline, citruline, creatinine, creatine, citrate, glutamine, glutamate, pyruvate, acetone, acetoacetate, acetate, GABA, lysine, arginine, alanine, lactate, isoleucine, 3-OH-butyrate. In order to enhance the identification of potential differences in biochemical composition caused by neuropathology, PLS-DA modeling was then employed, using knowledge of the specimen classification. Discriminant analysis maximizes any separation present between defined memberships classes (Y matrix) by appropriate weighting of the NMR variables used as the X matrix. In this case, we chose to analyze for systematic differences between ALS and control samples. The coefficients of the goodness of fit (R2) and prediction (Q2) were used to assess the model quality i.e. to establish whether any systematic differences between two groups of patients were present and the

81

variable importance in the projection computed by SIMCA-P were used to define which metabolites, if any, differentiated both groups. The best model consisted of two components (R2 = 0.645, Q2 = 0.33). It meant that the model fitted the data well but was not appropriate for prediction purposes. Validation test indicated that this model was valid for both ALS and control group. The most important parameters that contributed to class separation were signals from glutamate, histidine, and lactate as well as signals from acetate and alanine. Applying PLS-DA, the discrimination of ALS samples from the control samples was obtained (Fig. 1). In this model all patients from ALS group were classified correctly (100%)as opposite of only 12 from 15 patients from control group (80%).

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Conclusions A PLS-DA model was applied splitting the samples in two classes: normal samples formed class 1 and pathological samples formed class 2. All patients from ALS group were classified correctly and distinguished from control group on the basis of cerebrospinal fluid component analysis. Multivariate discriminant analysis performed on the two groups of CSF samples showed that it was possible to distinguish ALS patients from control patients. Because the coefficient of goodness of prediction for the model was not good, the further investigation on larger groups of patients was needed to confirm obtained results. In conclusion, 1H NMR data obtained from CSF samples can be treated with multivariate statistics to provide a useful screening tool. The technique is rapid and eventually could be used to detect other neuropathologies as well. The potential of the method should be further validated using greater number of samples and extended to include samples originating from patients suffering other diseases.

Reference List 1. Ellis,D.I., Dunn,W.B., Griffin,J.L., Allwood,J.W., and Goodacre,R. (2007) Metabolic

fingerprinting as a diagnostic tool, Pharmacogenomics. 8, 1243-1266. Acknowledgment: This work was supported by MSHE, Scientific Network 28/E-32/SN-0053/2007 28/E-32/SN-0053/2008.

82

STRUCTURAL STUDIES OF N-TERMINAL SEQUENCE OF DERMORPHIN BY MEANS OF SOLID STATE NMR

SPECTROSCOPY AND XRD

Katarzyna Trzeciak-Karlikowska1, Agata Jeziorna1, Grzegorz D. Bujacz2, Anna Bujacz2, Włodzimierz Ciesielski1, and Marek J. Potrzebowski1

1Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Łódź, Poland; 2Institute of Technical Biochemistry, Technical University of Łódź, Poland

Dermorphin is an opioid hepta-peptide (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) first

isolated from the skin of South American frogs belonging to the genus Phyllomedusa.

Dermorphin is about 30-40 times more potent than morphine but less likely to produce drug

tolerance and addiction. Synthetic analog of dermorphin containing L-alanine is biologically

inactive.

In this paper we present structural studies of two N-terminal sequences of dermorphin: Tyr-D-

Ala-Phe, Tyr-D-Ala-Phe-Gly and their L-alanine analogs Tyr-L-Ala-Phe, Tyr-L-Ala-Phe-Gly.

The application of 1D (CP/MAS) and 2D (PDSD, DAR and DCP/MAS) Solid State NMR

(SS NMR) experiments in spectral assignment of peptides under investigation will be

discussed. The comparative analysis of data obtained from SS NMR and X-ray

crystallography will be shown.

Figure. 2D 13C-13C Dipolar Assisted Recoupling (DAR) correlation of 13C, 15N labeled of tetrapetide Tyr-D-Ala-Phe-Gly

83

NEW PERMANENT MAGNETS FOR MRI

Krzysztof Turek1, Piotr Liszkowski1, and Boguslaw Tomanek2

1AMAG Sp. z o.o., Kraków, Poland; 2Institute of Nuclear Physics of Polish Academy of Science, Kraków, Poland

The main application of nuclear magnetic resonance is imaging (MRI) of a human body. AMAG Sp. o.o. has developed a number of permanent magnets for MRI systems, such as 0.08 T for MRI of hyperpolarized gases, 0.2 T for animal MRI, whole body 0.2 T 4-post magnet, 0.2 T “one side” cylindrical magnet and 0.2 T magnet based on Halbach concept. The latter two structures are dedicated for skin cancer and brain MRI respectively and can be sued for MRI using gradientless imaging method developed by the Institute for Biodiagnostics of National Research Council of Canada. The cylindrical magnet consists of 350 Nd-Fe-B blocks of the total weight 350 kg whereas the Halbach structure comprises about 500 Nd-Fe-B blocks and 700 non-magnetic elements of the total weight of about 1000 kg. The shape of the Halbach magnet reminds a typical cylindrical MRI superconducting magnet with the bore of 500 mm in diameter and 500 mm long. However the direction of the magnetic field is perpendicular to the magnet geometrical axis. The 0.1% homogeneity required for the gradientless method is obtained before final shimming in an elliptical cylinder of 185 (axis) x125 (axis) x 45 mm (length).

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ASSESSMENT OF CARDIAC FUNCTION RESERVE IN MURINE MODELS OF HEART FAILURE, IN VIVO STUDY

U. Tyrankiewicz13, T. Skorka1, S. Heinze-Paluchowska1, M. Jablonska1,2, M. Wozniak3,

L. Drelicharz3, and S. Chlopicki3

1Department of Magnetic Resonance Imaging, H. Niewodniczanski Institute of Nuclear Physics PAN, Krakow, Poland; 2Departament of Medical Physics and Biophysics, AGH University of Science and Technology, Krakow, Poland; 3Department of Experimental

Pharmacology, Chair of Pharmacology CMUJ, Krakow, Poland, Purpose:

The aim of the present work was to assess the alterations in systolic and diastolic cardiac function for better understanding of mechanisms leading to cardiomyopathy in two different murine models of the dilated cardiomyopathy: resulted in genetically induced heart hypertrophy and being pathology after oncological treatment.

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Fig.1. Normalized LV slice area over the time as measured from the R-wave. Ejection (ER) and Filling (FR) Rates are equal to absolute value of regression line slopes in units of 1/ms. Fractional Area Change (FAC) is equal to normalized difference between end-diastolic and end-systolic LV slice areas. Methods and Materials:

Cardiac MRI was used to assess the area of the short axis left ventricle crossection at the level of the papillary muscles. Various parameters have been deriven from the resulting area-time curve to characterize cardiac muscle function (Fig. 1). These were: HR (heart rate), ER (ejection rate), FR (filling rate) and FAC (fractional area change). Additionally, myocardial reserve was measured in response to pharmacologically induced (dobutamine) stress.

85

Two murine models of heart failure were used:

(1) Tgαq*44 mice (Tg mice) that develop heart failure (HF) during their lifespan [1,2]. These mice were observed at age of 4, 8, 9,12,13 and 15 months.

(2) Doxorubicin induced HF (DOX mice), C57BL/6 mice, 5 weeks of doxorubicin treatment 2 mg/kg i.p. injected twice weekly, which were observed 7 weeks after the beginning of chemotherapeutic treatment;

Experiments were performed on 4.7T/310 magnet (Bruker) system equipped with MARAN DRX (Resonance Instruments) console. All animal experimental procedures were approved by Ethic Commission of Jagiellonian University.

Results: In the control group, dobutamine stimulation improves chronotropic, inotropic and lusitropic reserve of LV (HR, ER and FR response, respectively). In murine heart pathology models however, ER and FR response was altered. In Tg mice FR response to dobutamine was not visible at age of 4 months but was relatively strong in age of 8-9 months, whereas at late stage of heart pathology the this reserve decreased dramatically. ER response to dobutamine stayed at approximately the same level despite of decreasing resting value. In DOX-treated mouse both ER and FR responses to pharmacologically induced stress was impaired.

Acknowledgments: This work was supported by PMSHE (grant NN 518419733) References: 1. Mende et al, JMCC (2001), 2. Drelicharz et al, BRC (2008) 3. Earls J. P., Ho V. B., et al, JMRI (2002) 4. Wiessman F., Ruff J., et al, Circ. Res. (2001)

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LOCAL AND COOPERATIVE DYNAMICS IN MODIFIED POLY(DIMETHYLSILOXANE)S STUDIED BY FAST FIELD CYCLING

NMR, BROADBAND DIELECTRIC AND FOURIER TRANSFORM INFRARED SPECTROSCOPIES

Wiktor Waszkowiaka, Aleksandra Wypycha, Maria Dobiesa, Stefan Jurgaa,

and Hieronim Maciejewskib.

aDepartment of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Poznań, Poland; bPoznan Science and Technology Park, Poznań, Poland

Poly(dimethylsiloxane) (PDMS) is an organic polymer made of silicon-oxygen chain with linked methyl groups. Unique properties of PDMS compared with other organic polymers make them very interesting class of substances. Although linear PDMS has already been widely studied, its grafted copolymers are still worth investigating due to their potential applications. The linear PDMS is partially crystalline, while structure of grafted copolymers depends on the type of a modifier. An incorporation of following side chains (a) (-CH2)3-O-CH2-(CHOCH2), (b) (-CH2)3-(OCH2CH2)7-OCH3 , (c) (-C16H33) to the backbone influences morphology and molecular dynamics of PDMS. It was found that PDMS copolymers grafted by side chain of type: (a) has amorphous structure, whereas an incorporation of the (b) and (c) modifiers leads to semicrystalline morphology formed by an arrangement of grafted long side chains.

Fast Field Cycling NMR (FFC) studies performed in room temperature suggest the Rouse dynamics for each investigated copolymers [1]. The Broadband Dielectric Spectroscopy (BDS), performed in wide temperature range allowed to detect various relaxation processes like: β relaxation - assigned to local motions, α relaxation - connected to segmental chain motions of polymer backbone, αc relaxation - coming from restricted chain mobility by crystalline phase.

In order to determine which part from investigated copolymers of type (b) and (c) crystallizes, the Fourier Transform Infrared spectroscopy (FTIR) studies were performed in broad temperature range. It was found that crystallization sensitive bands assigned to long alkyl or poly(ethylene oxide) chains had appeared in FTIR spectra below melting temperature [2]. It suggests that crystallization is driven by an arrangement of relatively long polymer chains grafted to PDMS backbone. Acknowledgements: This work is supported by 6th Framework Programme under SoftComp Grant No 502235-2. A. Wypych acknowledges the Ministry of Science and Higher Education in Poland for a Postdoctoral Fellowship (POL-POSTDOC III).

References: [1] R. Kimmich, E. Anoardo, Progress in Nuclear Magnetic Resonance Spectroscopy, 44

(2004) 257–320. [2] L. Sun, Y. Liu, L, Zhu, B. S. Hsiao, C. A. Avila-Orta, Polymer, 45 (2004) 8181–8193.

87

A NOVEL DETERMINATION OF 13C NUCLEAR MAGNETIC MOMENT IN TERMS OF THE 3He

FROM GASEOUS 13CH4 NMR SPECTRA

Marcin Wilczek1 , Anna Szyprowska2, and Włodzimierz Makulski1 1Laboratory of NMR Spectroscopy, Department of Chemistry, Warsaw University,

Warsaw, Poland; 2Pharmaceutical Research Institute, Warsaw, Poland

From the earlier atomic and molecular beam experiments it is known that NMR spectroscopy is well suited for a precise determination of magnetic properties like nuclear magnetic dipole moments. Traditional techniques as the static spectroscopy in homogenous magnetic fields can be applied. Measurements are conducted in bulk materials and always corrections for susceptibility and shielding effects are needed. These effects can entirely be omitted when gas phase studies are performed. Extrapolation of appropriate chemical shift values to the zero-pressure limit is the heart of the matter of this procedure [1]. Slightly modified version of this method exploits helium-3 atoms, magnetic moment of which is known with a high accuracy [2]. Very few new results received from gas phase NMR and theoretical calculations can be found in [3].

In this work we examined the 1H, 13C and 3He chemical shifts in gaseous mixtures of helium and methane molecules. The gaseous samples containing small amounts of 3He/13CH4 mixture in an excess of CO2 and Xe as buffers were prepared at total densities in the range 0.22 – 1.26 mol/L. The 1H, 13C and 3He NMR resonance frequencies were measured on a Varian INOVA 500 spectrometer at 300 K.

At a constant external magnetic field B0 = 11.7578 MHz the measured resonance frequencies are strictly linearly dependent on CO2 and Xe gas densities. This is consistent with the general approach used for the description of gas-phase NMR experiments. The Larmor precession frequencies observed, which are affected by intermolecular interactions, are as follows: ν0(

3He) = 381.3572155(2) MHz, ν0(13C) = 125.8762395(7) MHz

and ν0(1H) = 500.6068164(5) MHz. Recalculated from those data isotropic experimental

values in methane molecule are: σ0(1H) = 30.593(24) ppm, σ0(

13C) = 195.01(90) ppm in the absolute shielding scale. Experimental and theoretical shielding constants for helium and 1H and 13C nuclei in methane [4] were also used to receive 13C nuclear dipole moment for the free nuclei in its ground state. New experimental value µ(13C) is +0.70236943(63)µN, where µN is the nuclear magneton. This more precise quantity is in good agreement with previously reported results. REFERENCES: [1] A.Antušek, K.Jackowski, M.Jaszuński, W.Makulski,, M.Wilczek, Chem.Phys.Lett., 411 (2005) 111. [2] A.Rudziński, M.Puchalski, K.Pachucki, J.Chem.Phys., 130 (2009) 244102. [3] http://www.icho.edu.pl/PL/Zespoly/22/ [4] M.Jaszuński, K.Jackowski, Nuclear Magnetic Dipole Moments from NMR Spectra-Quantum Chemistry and Experiment, Lecture Notes in Physics, Springer: Berlin, 2008; Vol.745, p.233.

88

EPR EXAMINATION OF FREE RADICALS IN RADIATIVE STERILIZED PIPERACILLIN

Sławomir Wilczyński1, Barbara Pilawa1, Marta Ptaszkiewicz2, Janusz Swakoń2,

Paweł Olko2, Robert Koprowski3, and Zygmunt Wróbel3

1Medical University of Silesia in Katowice, School of Pharmacy and Laboratory Medicine,

Department of Biophysics, Sosnowiec, Poland; 2Department of Radiation Physics and Dosimetry, Institute of Nuclear Physics, Cracov, Poland; 3University of Silesia, Faculty

of Computer Science and Material Science, Institute of Computer Science, Sosnowiec, Poland

Free radicals in radiative sterilized piperacillin were studied by electron paramagnetic resonance (EPR) spectroscopy. Piperacillin sodium salt is the semi-synthetic antibiotics with broad-spectrum antibacterial activity and have good antibacterial activity against Gram-positive and-negative bacteria. [1]. Chemical structure of piperacillin is presented in Figure 1 [1]. Sterilization of the analyzed antibiotic was performed by gamma irradiation in THERATRON 780E containing isotope 60Co. Dose of gamma irradiation of 25 kGy was used.

First-derivative spectra were measured by an X-band (9.3 GHz) EPR spectrometer with modulation of magnetic field 100 kHz. Microwave power in the range of 0.7-70 mW was used. Free radical concentration was determined. Ultramarine and a ruby crystal were the references. Spin-spin and spin-lattice relaxation in the tested antibiotic were analyzed. Changes in paramagnetic centers system during storage of the drug was obtained. Effect of oxygen on free radicals in piperacillin during its storage was examined. Changes of integral intensity of EPR spectra of irradiated piperacillin with increasing time of storage were fitted by theoretical two-exponential function. Fig. 1. Chemical structure of piperacillin [1]. Fig. 2. EPR spectrum of gamma irradiated piperacillin. The spectrum was recorded after irradiation with microwave power of 2 mW.

References: [1] Chemia leków, A. Zejc, M. Gorczyca, PZWL Warszawa, 2002 Acknowledgements: This study was financially supported by Medical University of Silesia in Katowice (Grant no. KNW-2-156/09).

CH

NH

N

N

CO

O

O

C2H5

S

N

CO NH

O

CH3

CH3

COONa

NH

89

FREE RADICALS PROPERTIES OF DIETARY SUPPLEMENTS BASED ON

FISH AND PLANT OILS APPLIED IN COSMETOLOGY

Wilczyński Sławomir1, Zdybel Magdalena1, Barbara Pilawa1, Deda Anna1,2, and Pierzchała Ewa2

1Medical University of Silesia in Katowice, School of Pharmacy and Laboratory Medicine, Department of Biophysics, Sosnowiec, Poland; 2Medical University of Silesia in Katowice,

School of Pharmacy and Laboratory Medicine, Department of Esthetical Medicine, Katowice, Poland.

Free radicals play an important role in the pathogenesis of many diseases [1,2]. Some

of dietary supplement may have protective properties against free radicals. Preventive efficiency of dietary supplements depend on antioxidants contents [1,2]. To the most popular dietary supplements preparation containing plant and fish oils belong to. Antioxidative potential of selected dietary supplements was studied with the aid of electron paramagnetic resonance spectroscopy at X-band microwave frequencies (9.3 GHz). After adding to analyzed preparations the standard solution of free radicals (DPPH) the EPR spectra parameters were estimated. Amplitudes (A), linewidths (∆Bpp) and integral intensities (I) of the spectra were determined. Oenothera oil (OO), sardine fish oil (SFO) and two different samples of oli from greenland shark liver (OGS 1 and OGS 2) were analyzed.

All studied dietary supplements are effective in free radicals scavenging. In the decreasing free radicals scavenging capacity, the order was: OSG 1, SFO, OSG 2 and OO. No significant changes of linewidths were stated (tab. 1)

Table 1. Amplitudes of EPR lines (A) and linewidths (∆Bpp) for all studied samples.

Sample A [a.u.] ∆∆∆∆Bpp (mT) + 0.02

DPPH 1.60 0.13

OO 0.42 0.12

SFO 0.23 0.11

OGS1 0.17 0.12

OGS2 0.39 0.12

It was proven that EPR technique is suitable to antioxidative properties of the studied

dietary supplements determination. Obtain results may indicate that oils of animal origin are more efficient in free radicals scavenging in comparison with dietary supplements based on oils of plant origin.

References:

1. Bartosz G. Druga twarz tlenu. Wolne rodniki w przyrodzie. Warszawa: PWN, 2006. 2. Duda-Chodak A, Tarko T. Wolne rodniki – za i przeciw. Laboratorium 2007, 11: 48-50.

Acknowledgements: This study was financially supported by Medical University of Silesia in Katowice (Grant no. KNW-2-156/09)

90

RELAXATION PARAMETERS OF GD(III) AND MN(II) IONS IN WATER FROM THE PERSPECTIVE OF CONTRAST AGENTS

Milosz Wojciechowski and Danuta Kruk

Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Krakow, Poland

Paramagnetic systems are materials with positive magnetic susceptibility, associated

with unpaired electrons. The paramagnetic solutions of interest for Magnetic Resonance

Imaging contain transition metal complexes. The presence of unpaired electron spins has a

profound influence on Magnetic Resonance Spectra of such solutions. The origin of the

effects is found in the large value of the electronic magnetic moment about 650 times that of

the proton. The paramagnetic species enhance the nuclear spin relaxation rates. The

paramagnetic relaxation enhancement is caused by random variations of the electron spin –

nuclear spin dipole-dipole interactions, which open a pathway for the nuclear spin relaxation.

A very active field of applications of the paramagnetic relaxation enhancement effects is the

development and use of paramagnetic materials as contract agents in Magnetic Resonance

Imaging. In this work electron spin interactions and relaxation parameters of Gd(III) and Mn(II)

ions are discussed in the contexts of their further effects on proton relaxation of the solvent

molecules. Proton relaxation has been measured at low and high magnetic fields for solutions

of increasing viscosity slowing down reorientational motion of the paramagnetic complexes.

Effects of the molecular tumbling on the paramagnetic relaxation enhancement are

investigated in detail.

This work has been financed by Polish Ministry of Science and Education, grant No N N202 105936

91

UNIPLANAR GRADIENT COILS – FROM DESIGN TO APPLICATI ONS

Grzegorz Woźniak*, Tomasz Skórka*, Krzysztof Jasiński*, Tomasz Banasik*, Mohammad Mohammadzadeh**, Dominik von Elverfeldt**, Jürgen Hennig**, and Władysław P. Węglarz*

*IFJ PAN Kraków, Poland; **Freiburg University, Freiburg, Germany

Cylindrical gradient coils typically used for MRI imaging have limitations in gradient

strength as well as restrictions in access to sample. In recent years growing interest in coils with different geometry, allowing for better sample access and/or higher local magnetic field gradient, is observed [1]. Aim of this study was to design, build and test flat gradient coil set for 3D micro imaging. Comparison of theoretical results with simulated parameters and physical tests using 4.7 T and 9.4 T MRI scanners are presented. Flat gradient coil set was designed using stream function approach [2,3]. Coil size was 10 by 10 cm and the assumed field of view (FOV) 1.5x0.15x1.5cm with centre 5.5 mm above coil surface. The design procedure has been implemented using scripting language of the Comsol Multiphysics package (Comsol AB, Sweden). Magnetic field generated by current patterns were analysed using Comsol AC/DC module to verify theoretical predictions. In order to compensate for distortions introduced by bounding wires and connection paths, an additional current paths were considered. Temperature simulations were done to assess requirement for cooling method, thermal conductivity of materials and limits of the current in the coils. Theoretical, simulated and physical values of coils parameters were compared on a manufactured prototype coil set. Coils set has been constructed and tested. Overall achieved FOV size and gradient strength was in agreement compared to original theoretical calculations. Temperature measurements show that energy deposit can by received by water cooling system at 150C providing temperature below 500C with 50A power supply and 20% filling factor at all three coils on. Results of the temperature distribution of the probehead surface is presented. Distributions of magnetic field along work cross-section are presented. MR images of the test sample containing set of parallel rows filled with water shows regions of gradient linearity. Results confirm feasibility of manufacturing effective flat gradient coils for MR micro-imaging as well as usefulness of simulation which allow to determine the best way in every step of design. Future tests are prepared. References: 1.see recent ISMRM,e.g.posters #3055-3074,2008,Honolulu,Hawaii,US. 2.R.Lemidasov,R.Ludwig,Conc.Magn.Reson.B.,Vol. 26B(1)(2005)p.67–80. 3.G.Wozniak,T.Skorka,K.Jasinski,W.P.Weglarz,2008,NMR School, Wierzba,Poland Acknowledgments: This work was supported by European Community grant FP6-2004-NEST-C-1-028533

92

Dendrimer-like star-branched polystyrene

DYNAMICS OF POLYSTYRENE OF DIFFERENT MOLECULAR ARCHITECTURE AS REVEALED BY NUCLEAR MAGNETIC RESONANCE, BROADBAND DIELECTRIC SPECTROSCOPY

AND REOLOGY

Aleksandra Wypycha, Monika Makrocka-Rydzyka, Grzegorz Nowaczyka, Eugeniusz Szcześniaka, Stefan Jurga*a, Akira Hiraob, and Takumi Watanabeb.

a Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University,

Poznań, Poland; b Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo Japan

Dendrimer-like star-branched polymers have recently appeared as a new class of hyperbranched polymers synthesized by means of living anionic polymerization [1]. Their structure with chain units branching out from a center cord comprises several layers (so-called generations).They possess interesting properties resulting from unusual architectural features obtained by methods of controlled polymerization [2]. However, they require a detailed characterization on a molecular level, what can be realized using modern spectroscopic methods. The aim of this study is to determine the influence of the system architecture on molecular dynamics in linear and dendrimer-like star-branched polystyrenes.

The analysis of temperature behavior of: 13C NMR, Broadband Dielectric

Spectroscopy and Rheological data indicates an existence of three motional processes denoted as α, β and γ in order of decreasing temperature. The α relaxation is related to the dynamics of glass transition, the β -relaxation was assigned to a motion of the phenyl rings, while the γ relaxation was attributed to the local motions of the methylene units (present due to tail-to-tail or head-to-head sequences). A broadening of the distribution of segmental relaxation times in the polymer of dendritic structure, with respect to the linear one, was observed.

References: [1] W.A. Braunecker, K. Matyjaszewski, Prog. Polym. Sci., 32, 93, 2007. [2] B.Lepoittevin, R. Matmour, R. Francis, D.Taton, Y.Gnanou, Macromolecules, 38, 3120,

2005. [3] A. Deffieux, M. Schappacher, A. Hirao, T. Watanabe, J. Am. Chem. Soc., 130 (17), 5670,

2008. Acknowledgements: This work is supported by 6th Framework Programme under SoftComp Grant No 502235-2 and research grant No N N202 128536 (Poland). A. Wypych acknowledges the Ministry of Science and Higher Education in Poland for a Postdoctoral Fellowship (POL-POSTDOC III).

93

SPECTRA OF HIGH DIMENSIONALITY FOR EASY RESONANCE ASSIGNMENT IN PROTEINS

Anna Zawadzka-Kazimierczuk, Krzysztof Kazimierczuk, and Wiktor Koźmiński

Faculty of Chemistry, University of Warsaw, Warsaw, Poland

Multidimensional experiments, invaluable in case of crowded spectra, may sometimes not ensure satisfactory results, when performed in a conventional way, where the evolution time space is sampled at equal intervals. The necessity of fulfilling the Nyquist Theorem in this case causes that having limited experimental time one has to compromise between resolution and spectral width. In such situation, when peaks overlap, it is hard to precisely evaluate chemical shifts. We have proposed the method which allows overcoming this problem. Random sampling of evolution time space, followed by Multidimensional Fourier Transform, provides spectra of very high resolution (it is possible to obtain natural linewidths) and enables performing experiments of high dimensionality (4D, 5D, 6D). The processing of such spectra takes advantage of resonance positions form spectra of lower dimensionality and thus results with a set of 2D spectra, which makes it very convenient to use. We propose several novel techniques which allow easy assignment of protein backbone signals. The experiments were performed on proteins of various sizes: ubiquitin (76 residues), protein interacting with NIMA-kinase from Cenarcheaum symbiosum (96 residues) and maltose binding protein (370 residues). References: [1] Zawadzka-Kazimierczuk A, Kazimierczuk K, Koźmiński W

J. Magn. Reson. (in press, doi: 10.1016/j.jmr.2009.10.006)

94

EFFECT OF TEMPERATURE ON EPR SPECTRA OF DOPA-MELANIN-NETILMICIN COMPLEXES

Magdalena Zdybel1, Barbara Pilawa1, Ewa Buszman2, Dorota Wrześniok2,

Ryszard Krzyminiewski3, Zdzisław Kruczyński3, Robert Koprowski4, and Zygmunt Wróbel4

1, 2Medical University of Silesia in Katowice, School of Pharmacy and Division of Laboratory Medicine, 1Department of Biophysics, Sosnowiec, Poland; 2Department of Pharmaceutical

Chemistry, Sosnowiec, Poland; 3Adam Mickiewicz University, Institute of Physics, Department of Medical Physics, Poznań, Poland; 4University of Silesia, Faculty of Computer

Science and Material Science, Institute of Computer Science, Sosnowiec, Poland

Types of paramagnetic centers in DOPA-melanin and its complexes with netilmicin were analysed by EPR spectroscopy. Free radicals with spin S = 1/2 and biradicals with spin S =1 were found in the studied melanin samples. Correlations between the integral intensities (I) of the EPR spectra and the measuring temperature (T) in the range 100-300 K were searched. Intensities of all the recorded EPR lines do not change with temperature according the Curie law. Numerical fitting of the experimental data by theoretical functions indicates that sum of functions for free radicals and biradicals are the best results for the obtained correlations of I = f(T). Intensities of EPR lines of free radicals fulfill the Curie law (I = C/T). The other function describes correlation between intensity of EPR lines of biradicals and temperature. DOPA-melanin was obtained according to Binn’s method [1]. Concentrations of complexing agent - netilmicin was 1x10-3 M. Measurements were done by the use of an X-band (9.3 GHz) spectrometer of BRUKER Firm. The first derivative EPR spectra were measured with low microwave power of 0.3 mW to avoid microwave saturation of line. Changes of amplitude (A), linewidth (∆Bpp), and integral intensity (I) of EPR spectra with the measuring temperature (T) were determined. Experimental data IT = f(T) were numerically analysed. The experimental product IT = f(T) was fitted by sum of the theoretical functions for free radicals and biradicals according to the formula [2-3]:

IT = C + D/(3 + exp(J/kT))

where: I – integral intensity of EPR line, T – the measuring temperature, and C – Curie constant, D – constant, J – energy of singlet-triplet excitation, and k – Boltzmann constant. The parameters C and D, and the energy of singlet-triplet excitation J, were calculated. The error of the fitting was calculated as follow:

δ = 1/N (ΣT| IT – IT(S)|)

where: IT – experimental product, IT(S) – theoretical product, N – the number of the experimental points. The Labview and the program for spectral analysis of Jagmar Firm (Kraków, Poland) were used to determination of the parameters of the EPR spectra. Integral The Matlab 7.20, Signal Processing and Statistics were used during the theoretical fitting of the experimental data. Free radicals and biradicals in melanin samples were earlier found by Kozdrowska et al. [4-5]. Free radicals (S = 1/2) and biradicals (S = 1) form complex paramagnetic centers system in DOPA-melanin and complexes of DOPA-melanin with kanamycin and Cu(II) [4-5].

95

References: [1] Binns F, Chapman RF, Robson NC, Swan GA, Waggot A. J Chem Soc C 1970: 1128-

1134. [2] Wertz JE, Bolton JR. New York: Academic Press; 1986. [3] Hatfield WE. [In:] Boudreaux EA, Mulay LN. New York, London, Sydney, Toronto: John

Wiley & Sons; 1976: pp. 357-359. [4] Kozdrowska L. Rozprawa doktorska. Uniwersytet Zielonogórski, Zielona Góra, 2005. [5] Kozdrowska L, Pilawa B, Świątkowska L, Buszman E, Wrzesniok D, Grzegorczyk A,

Więckowski AB, Wojtowicz W, Wilczok T. Abstracts of 21st International Meeting on Radio- and Microwave Spectroscopy RAMIS 2005. Poznań-Będlewo 2005, p. 33.

[6] Pilawa B, Latocha M, Krzyminiewski R, Kruczyński Z, Buszman E, Wilczok T. Phys Med 2004; 1: 96-98.

Acknowledgements: This study was financially supported by Medical University of Silesia in Katowice (Grant no: KNW- 2-126/09).

96

SPIN-LATTICE RELAXATION IN THERMALLY STERILIZED CHLORTALIDONE

Magdalena Zdybel, Jakub Adamczyk, Barbara Pilawa, Magdalena Kościelniak,

and Daria CzyŜyk

Medical University of Silesia in Katowice, School of Pharmacy and Laboratory Medicine, Department of Biophysics, Jedności 8, 41-200 Sosnowiec, Poland,

Spin-lattice relaxation in Chlortalidone was studied by electron paramagnetic resonance (EPR). Chlorthalidone is a diuretic drug used to treat hypertension. [1]. Chemical structure of Chlortalidone is shown in Figure 1 [1]. Chlortalidone was sterilized according to the valid norms [2] in hot air oven at the following conditions: 160 oC by 120 minutes, 170 oC by 60 minutes, and 180 oC by 30 minutes. The aim of this work was to determine effect of conditions of thermal sterilization on spin-lattice relaxation in Chlortalidone. Changes of spin-lattice interactions in thermally sterilized drug point out that its chemical structure are modified. Examination of magnetic interactions in drug is helpful to obtain optimal condition of its thermal sterilization.

NH

O

OH

Cl

SO

ONH2

Fig. 1. Chemical structure of Chlortalidone.

Thermally sterilized Chlortalidone is paramagnetic. EPR spectra were measured for Chlortalidone heated at 160 oC, 170 oC and 180 oC. The spectra were recorded by the use of EPR spectrometer produced by RADIOPAN Firm (Poznań). Magnetic modulation was 100 kHz. Microwave frequency from X-band (9.3 GHz) was measured by MCM 101 recorder of EPRAD Firm (Poznań). EPR spectra were measured in the wide range of microwave power from 0.7 mW to 70 mW. Continuous microwave saturation of EPR lines was applied to examination of spin-lattice relaxation processes [3-5].

References: [1] ZEJC A., GORCZYCA M. (RED.) Chemia leków, PZWL, Warszawa, 2002 [2] Farmakopea VII [3] STANKOWSKI J., HILCZER W. Wstęp do spektroskopii rezonansów magnetycznych. PWN, Warszawa, 2005 [4] WERTZ J. E., BOLTON J. R. Electron Spin Resonance Theory and Practical Applications. New York, London 1986 [5] EATON G. R., EATON S. S., SALIKHOV K. M. (Eds.). Foundations of modern EPR, World Scientific, Singapore, New Jersey, London, Hong Kong 1998 Acknowledgements This study was supported by Medical University of Silesia in Katowice (Grant no: KNW 1-142/09).

97

DISPERSION MEASUREMENTS OF T1ρ IN PROTEIN SOLUTIONS

Łukasz Żelazny1, Dorota Wierzuchowska2, and Barbara Blicharska1

1Jagiellonian University, Department of Physics, Krakow, Poland;

2Pedagogical University, Institute of Physics, Kraków, Poland

Relaxation time measurements in rotating frame and its dependence on frequency, called NMR dispersion, may be a source of information about molecular dynamics and structure changes in aqueous protein solution. Assuming a pure dipole-dipole interaction as a dominant relaxation mechanism of proton in free and bounded water we are able to calculate the correlation time from T1ρ dispersion profile (that is dependence of relaxivity in rotating frame R1ρ = 1/T1ρ on Β1).

These calculations were done for samples of different protein solution ( albumin and lysozyme) at different protein concentration. The changes of molecular dynamics, which appear after thermal (irreversible) or alcohol (reversible) denaturation, are discussed.

98

THE ONE NMR™ PROBE FROM VARIAN AND THEIR COMBINATION TOOLS

Thomas Zellhofer

Varian Deutschland GmbH, Darmstadt, Germany

The OneNMR™ Probe represents the most significant advancement in solution-state probe technology in over a decade. The OneNMR probe uses a hybrid approach to deliver the performance advantages of both the classic "carbon probe" (direct detect probe) and the highly sensitive "proton probe" (indirect detect probe). It is simultaneously optimized for both high and low band frequencies in a single unit. Increased 1H sensitivity is just one component of this new technology, it also provides excellent RF homogeneity, excellent salt and solvent tolerance, excellent water suppression, and excellent lock sensitivity compared to traditional probe technology. This combination offers unprecedented performance and flexibility for all your routine NMR experiments.

The OneNMR probe provides you with all of the data required for routine structure analysis of organic molecules, significantly reducing the labor and productivity-loss associated with switching probes for multiple experiments.

The flexibility and power of the OneNMR probe products is most pronounced when combined with automation accessories and VnmrJ 3. This latest release of VnmrJ was redesigned with your workflow in mind. This combination (VnmrJ 3, OneNMR probe, ProTune, and Autosampler) are the perfect solution for all you routine NMR needs.

99

NUCLEAR RELAXATION IN SELECTED IMIDAZOLIUM COMPOUNDS: EFFECT OF QUADRUPOLAR INTERACTIONS

Włodzimierz Masierak1, Cezary Uniszkiewicz1, Alexiei Privalov2, Franz Fujara2, Wojciech

Medycki3, and Danuta Kruk4

1 Institute of Physics, Bydgoszcz University, Poland; 2 Technische Universität Darmstadt,

Germany; 3Institute of Molecular Physics, PAS , Poznań, Poland; 4Institute of Physics,

Jagiellonian University, Krakow, Poland

Imidazolium compounds are very interesting example of systems of mutually coupled

dipolar and quadrupolar spins. Such systems contain several types of dynamically non-

equivalent imidazolium rings. As a consequence of the different dynamics one experimentally

observes proton relaxation as well as polarization transfer effects. Polarization transfer can

take place only if there is an efficient (not averaged out) coupling between the dipolar and

quadrupolar spins. If the magnetic field is set to a value which leads to the Zeeman splitting

of the dipolar spin matching the energy splitting of the quadrupolar spin (determined by the

quadrupolar and Zeeman interactions), the dipole-dipole coupling causes the polarization

transfer. The mutual dipole-dipole coupling links transitions of the dipolar spin to some

transitions of the quadrupolar spin, so they cannot occur independently. The polarization of

the dipolar spins is transferred to the quadrupolar subsystem with an efficiency directly

related to the probability of the joint transitions. The fact that one can clearly see the

polarization transfer effects (as dips of the magnetization curve recorded versus the magnetic

field) allows us to state that some of the imidazolium rings are quite rigid and therefore there

are efficient proton – quadrupolar nuclei dipole – dipole couplings. At the some time proton

relaxation is observed. We attribute this process to a fast movement of protons belonging to

other rings. The relaxation processes are considerably affected by dipole-dipole couplings to

the quadrupolar nuclei.

This work has been financed by Polish Ministry of Science and Education, grant No N N202

172135

100

APPLICATION OF FAST FIELD CYCLING NMR RELAXATION TO SELECTED IMIDAZOLIUM COMPOUNDS

Cezary Uniszkiewicz1, Włodzimierz Masierak1,2, Alexiei Privalov3, Franz Fujara3, Wojciech

Medycki4, and Danuta Kruk5

1 Institute of Physics, Bydgoszcz University, Bydgoszcz, Poland; 2 School of Economy,

Bydgoszcz, Poland; 3 Technische Universität Darmstadt, Germany; 4Institute of Molecular

Physics, PAS , Poznań, Poland; 5 Institute of Physics, Jagiellonian University,

Krakow, Poland

This work is concerned with several aspects of field dependent relaxation processes

and polarization transfer effects in complex solid state systems containing mutually coupled

quadrupolar and dipolar spins (of the spin quantum number ½). Dipole - dipole interactions

provide the coupling between the two spin subsystems.

These phenomena are illustrated by several experimental examples for various

imidazolium compounds. Such systems are very attractive because of variety of effects caused

by interplay between quadrupolar and dipolar interactions carrying unique information on

molecular dynamics.

This work has been financed by Polish Ministry of Science and Education, grant No N N202

172135

101