i
VASCULAR BIOCHEMISTRY OF VITAMIN B12: EXPLORING THE RELATIONSHIP BETWEEN INTRACELLULAR COBALAMIN AND REDOX STATUS IN HUMAN AORTIC ENDOTHELIAL CELLS
A dissertation submitted
to Kent State University in partial
fulfillment of the requirements for the
degree of Doctor of Philosophy
by
Edward Suarez Moreira
May, 2010
i
Dissertation written by
Edward Suarez Moreira
B.Sc., Universidad de la República, Uruguay 2002
Ph.D. Kent State University, 2010
Approved by
______, Chair, Doctoral Dissertation Committee
Nicola E. Brasch, Ph.D.
______, Co-Adviser, Doctoral Dissertation Committee
June Yun, Ph.D.
______, Member, Doctoral Dissertation Committee
William M. Chilian, Ph.D.
______, Member, Doctoral Dissertation Committee
Roger B. Gregory, Ph.D.
______, Graduate Faculty Representative
Eric M. Mintz, Ph.D.
Accepted by
______, Chair, School of Biomedical Sciences
Robert V. Dorman, Ph.D.
______, Dean, College of Arts and Sciences
John R. D. Stalvey, Ph.D.
ii iii
TABLE OF CONTENTS
LIST OF FIGURES ...... VII
LIST OF TABLES ...... X
LIST OF ABBREVIATIONS ...... XI
DEDICATION...... XV
ACKNOWLEDGEMENTS ...... XVI
ABSTRACT ...... XIX
CHAPTER 1 INTRODUCTION ...... 1
1.1 Cobalamin Structure and Catalytic Function ...... 1
1.1.1 Cobalamin Structure ...... 2
1.1.2 Catalytic Function ...... 6
1.2 Cobalamin Metabolism in Mammals ...... 10
1.2.1 Transport and Processing ...... 10
1.2.2 Cobalamin Deficiency and its Diagnosis ...... 18
1.3 Cobalamin and Reactive Species ...... 20
1.3.1 Reactive Oxygen and Nitrogen Species ...... 20
1.3.2 Cobalamin and Reactive Species ...... 23
1.4 Cobalamin and Vascular Biology ...... 25
1.4.1 The Vascular Endothelium ...... 25
1.4.2 Oxidative Stress and Cardiovascular Disease ...... 26
1.4.3 Cobalamin and Hyperhomocysteinemia ...... 28
1.5 Non-cofactor Functions of Cobalamin ...... 30
iii
1.6 Aims ...... 31
CHAPTER 2 SYNTHESIS AND CHARACTERIZATION OF BIOLOGICALLY
RELEVANT COBALAMINS ...... 33
2.1 Introduction ...... 33
2.2 Experimental Section ...... 37
2.2.1 Materials ...... 37
2.2.2 General Methods ...... 37
2.2.3 Synthesis of Thiolatocobalamins ...... 39
2.2.4 Synthesis of Non-thiolatocobalamins...... 41
2.2.5 Kinetic Measurements ...... 44
2.3 Results ...... 45
2.3.1 Synthesis and Characterization of D,L-Homocysteinylcobalamin, N-Acetyl-
L-cysteinylcobalamin (Na+ salt) and 2-N-acetylamino-2-carbomethoxy-L-
ethanethiolatocobalamin...... 45
2.3.2 Acid-catalyzed Decomposition of GSCbl and NACMECbl ...... 46
2.3.3 Synthesis and Characterization of Sulfitocobalamin ...... 50
2.4 Discussion ...... 52
CHAPTER 3 EXPLORING THE ROLE OF GLUTATHIONE IN COBALAMIN
PROCESSING IN HUMAN AORTIC ENDOTHELIAL CELLS ...... 56
3.1 Introduction ...... 56
3.2 Experimental Section ...... 60
3.3 Results ...... 65
iv
3.3.1 Extraction and Identification of Intracellular Cbl Derivatives in HAEC ...... 65
3.3.2 Extraction and Identification of Intracellular Cbl Derivatives in HAEC in the
Presence of a Free Ligand Trap...... 72
3.3.3 Determination of Intracellular Cobalamins in GSH-depleted HAEC ...... 81
3.4 Discussion ...... 85
CHAPTER 4 VITAMIN B12 PROTECTS AGAINST HOMOCYSTEINE-
INDUCED CELL INJURY IN HUMAN AORTIC ENDOTHELIAL CELLS
...... 90
4.1 Introduction ...... 90
4.2 Experimental Section ...... 92
4.3 Results ...... 94
4.3.1 Cobalamin Protection against Homocysteine-Induced Increase in ROS ...... 94
4.3.2 Subcellular Localization and Cobalamin Protection against Homocysteine-
•- Induced O2 increase ...... 97
4.3.3 Cobalamin Protection against Homocysteine-Induced Cell Death ...... 100
4.4 Discussion ...... 105
CHAPTER 5 VITAMIN B12 AND REDOX HOMEOSTASIS: COB(II)ALAMIN
REACTS WITH SUPEROXIDE AT RATES APPROACHING
SUPEROXIDE DISMUTASE ...... 111
5.1 Introduction ...... 111
5.2 Experimental Section ...... 113
5.2.1 General Methods ...... 113
v
5.2.2 Kinetic Measurements ...... 116
5.2.3 Intracellular Studies...... 118
5.3 Results ...... 119
•- 5.3.1 In vitro Kinetic Studies on the Reaction between Cbl(II) and O2 ...... 119
•- 5.3.2 Cobalamin Protects against Intracellular O2 ...... 131
5.4 Discussion ...... 135
CHAPTER 6 SUMMARY AND FUTURE DIRECTIONS ...... 138
6.1 Summary ...... 138
6.2 Future Directions ...... 139
6.2.1 Glutathionylcobalamin and Cobalamin Processing...... 139
•- 6.2.2 Cobalamin as a Direct O2 Scavenger ...... 140
6.2.3 Cobalamin as General Antioxidant...... 141
REFERENCES ...... 143
vi
LIST OF FIGURES
CHAPTER 1 INTRODUCTION
Figure 1.1 Structure of the three tetrapyrrolic macrocycles ...... 3
Figure 1.2 Structures of naturally occurring cobalamins...... 4
Figure 1.3 Reaction scheme for the MeCbl-dependent methionine synthase
methylation of Hcy ...... 9
Figure 1.4 Reaction scheme for the isomerization catalyzed by methymalonyl-CoA
mutase ...... 12
Figure 1.5 Diagram of Cbl metabolism ...... 15
Figure 1.6 Production and detoxification of reactive oxygen species...... 21
Figure 1.7 Homocysteine metabolic pathways ...... 29
CHAPTER 2 SYNTHESIS AND CHARACTERIZATION OF BIOLOGICALLY
RELEVANT COBALAMINS
Figure 2.1 Structures of the thiols used for thiolatocobalamin synthesis ...... 36
Figure 2.2 UV/visible spectra of thiolatocobalamins ...... 42
Figure 2.3 1H NMR spectra of thiolatocobalamins ...... 43
Figure 2.4 Acid-catalyzed decomposition of thiolatocobalamins ...... 48
Figure 2.5 Kinetic traces for the acid-catalyzed decomposition of RSCbls ...... 49
Figure 2.6 Plots of kobs vs pH for the decomposition of RSCbls ...... 51 vii
Figure 2.7 Characterization of sulfitocobalamin ...... 53
CHAPTER 3 EXPLORING THE ROLE OF GLUTATHIONE IN COBALAMIN
PROCESSING IN HUMAN AORTIC ENDOTHELIAL CELLS
Figure 3.1 Key metabolic pathways involved in the synthesis of GSH ...... 59
Figure 3.2 GSCbl recovery ...... 67
Figure 3.3 Cbl uptake by HAEC ...... 69
Figure 3.4 Cobalamin profile in HAEC ...... 70
Figure 3.5 Representation of β-axial ligand exchange reactions ...... 73
Figure 3.6 Intracellular and artifactual pathways for GSCbl formation ...... 75
Figure 3.7 Artifactual formation of Cbl derivatives in HAEC ...... 76
Figure 3.8 Representation of the free ligand trap methodology ...... 78
Figure 3.9 Cobalamin profile in HAEC in the presence of a ligand trap ...... 80
Figure 3.10 Intracellular L-Cys and GSH concentration for HAEC in response to
L-Cys starvation as a function of time ...... 83
Figure 3.11 Cobalamin profile in GSH-depleted HAEC ...... 84
CHAPTER 4 VITAMIN B12 PROTECTS AGAINST HOMOCYSTEINE-
INDUCED CELL INJURY IN HUMAN AORTIC ENDOTHELIAL CELLS
Figure 4.1 L-Hcy-induced ROS increase ...... 95
Figure 4.2 L-Hcy specificity for eliciting ROS ...... 96
Figure 4.3 Effect of exogenous CNCbl on intracellular Cbl content ...... 98
Figure 4.4 Cbl protection against L-Hcy-induced oxidative stress ...... 99
Figure 4.5 Cbl protection against L-Hcy induced superoxide production ...... 102
viii
Figure 4.6 Subcellular localization of Hcy-induced oxidative stress ...... 103
Figure 4.7 Cbl protection against L-Hcy-induced cell death ...... 104
Figure 4.8 Cbl protection against L-Hcy-induced apoptosis ...... 106
CHAPTER 5 VITAMIN B12 AND REDOX HOMEOSTASIS: COB(II)ALAMIN
REACTS WITH SUPEROXIDE AT RATES APPROACHING
SUPEROXIDE DISMUTASE
•- Figure 5.1 O2 production by xanthine oxidase ...... 115
Figure 5.2 Cob(II)alamin oxidation by superoxide ...... 120
Figure 5.3 Cob(II)alamin reacts with H2O2 ...... 121
Figure 5.4 Catalase decreases the initial rate of Cbl(II) oxidation ...... 122
Figure 5.5 Competition kinetics with Cu,Zn-SOD ...... 126
Figure 5.6 Cbl(II) reacts directly with ferricytochrome c ...... 130
Figure 5.7 Effect of Cbl on paraquat-induced cell death ...... 134
ix x
LIST OF TABLES
CHAPTER 2 SYNTHESIS AND CHARACTERIZATION OF BIOLOGICALLY
RELEVANT COBALAMINS
Table 2.1 UV/visible and 1H NMR spectroscopy data for cobalamins ...... 47
CHAPTER 3 EXPLORING THE ROLE OF GLUTATHIONE IN COBALAMIN
PROCESSING IN HUMAN AORTIC ENDOTHELIAL CELLS
Table 3.1 Percentages of cobalamins extracted from HAEC in the absence or
presence of an added free ligand trap in the cell lysis buffer, and after GSH
depletion by L-Cys starvation ...... 71
CHAPTER 5 VITAMIN B12 AND REDOX HOMEOSTASIS: COB(II)ALAMIN
REACTS WITH SUPEROXIDE AT RATES APPROACHING
SUPEROXIDE DISMUTASE
•- Table 5.1 Experimental and simulated rates of Cbl(II) oxidation by O2 in the
presence of SOD ...... 129
•- Table 5.2 Second-order rate constants for the reaction of various O2 scavengers
•- with O2 ...... 132
x
LIST OF ABBREVIATIONS
AdoCbl adenosylcobalamin
BAEC bovine aortic endothelial cells
BHMT betaine homocysteine methyl transferase
Cbl cobalamin
Cbl(I) cob(I)alamin
Cbl(II) cob(II)alamin
Cbl(III) cob(III)alamin
CBS cystathionine beta synthase
CNCbl cyanocobalamin
CNS central nervous system
CVD cardiovascular disease
Cys cysteine
DCF dichlorofluorescein
DCFA dichlorofluorescein acetate
DMB 5,6-dimethylbenzimidazole
DNA deoxyribonucleic acid
DPBS Dubellco’s phosphate buffer saline e- electron
xi
EDTA ethylenediaminetetraacetic acid
ELISA enzyme linked immuno sorbent assay
ES-MS electrospray mass spectrometry
GSCbl glutathionylcobalamin
GSH reduced glutathione
GSSG oxidized glutathione
GPx-1 glutathione peroxidase 1
+ H2OCbl aquacobalamin
HAEC human aortic endothelial cells
HC haptocorrin
Hcy homocysteine
HMox heme oxygenase
HOCbl hydroxocobalamin
HPLC high performance liquid chromatography
HTL homocysteine thiolactone
IF intrinsic factor
LDL low density lipoprotein
MeCbl methylcobalamin
MES 2-(N-morpholino)ethanesulfonic acid
Met methionine
MeTHF methyl-5’-tetrahydrofolate
MCDB Modified Czapek Dox Broth
xii
MMA methylmalonic acid
MMACHC methylmalonic aciduria and homocystinuria type C
MMM methylmalonyl-CoA mutase
MnTEPyp MnIIImeso-tetrakis(o-N-ethylpyridinium-2’-yl)porphyrin
MS methionine synthase
MTT dimethyl thiazolyl diphenyl tetrazolium salt
NACCbl N-acetylcysteinylcobalamin
NACMECbl 2-N-acetylamino-2-carbomethoxy-ethanethiolatocobalamin
NADPH nicotinamide adenine dinucleotide phosphate, reduced form
NF-κB nuclear factor κ B
NMR nuclear magnetic resonance
NO2Cbl nitrocobalamin
NOCbl nitroxylcobalamin
NOS nitric oxide synthase
NOX NADPH oxidase
PBS phosphate buffer saline
RNS reactive nitrogen species
ROS reactive oxygen species
RSCbl thiolatocobalamin
SAM S-adenosylmethionine
SAH S-adenosylhomocysteine
SO3Cbl sulfitocobalamin
xiii
SOD superoxide dismutase
TC transcobalamin
THF tetrahydrofolate
TNF-α tumor necrosis factor α
XO xanthine oxidase
xiv xv
DEDICATION
To mom, dad, Risel, and Sofía
(a mamá, papá, Risel y Sofía)
In loving memory of:
Antonio Mario Moreira (1911-2005) and Margarita Ibaldi (1920-2008)
xv xvi
ACKNOWLEDGEMENTS
First and foremost I would like to express my gratitude to my advisers Dr. Nicola
Brasch and Dr. June Yun. Over the past six years they have guided me, taught me,
advised me and overall made me a much better scientist. They have encouraged me to
think independently and have always been open to new ideas and productive scientific
discussion. They have always believed that I could succeed and encouraged me to go on
at times when the path to the Ph.D. seemed too steep and almost impossible to walk.
I would also like to express my gratitude to the members of my dissertation
committee Dr. Roger Gregory and Dr. William Chilian for offering their help and advice.
I would also like to thank Dr. Eric Mintz for being the faculty representative for my oral
defense.
The journey to academic success is not just academic. Therefore I would like to
warmly thank the administrative personnel at the Kent State Department of Chemistry,
Janie Viers, Diana Skok, Scott Cornell and Erin Michael, and the administrative
personnel at NEOUCOM Department of Integrative Medical Sciences, Carolyn Miller
and Karen Greene.
Luckily we do not travel alone on our way to the doctorate. We share ups and
downs, laughter and frustration with our fellow graduate students. Thanks to my labmates past and present at the Brasch lab. Special thanks to Mike Radomsky, Riya Mukherjee
xvi
and Dr. Edward Donnay. I would also like to thank the graduate students and research staff at NEOUCOM and especially Danielle Speicher.
During my doctoral research I had the privilege to work in different labs learning from different mentors in different environments. I would like to thank Dr. Donald
Jacobsen for the opportunity to work in his lab at the Cleveland Clinic. The experience was educational. Special thanks to Dr. Patricia Dibello and Dr. John Barbato for all their technical help, support and encouragement while I was at the Cleveland Clinic.
My training years at the University of Uruguay gave me the tools I needed to pursue a doctoral degree. I would especially like to express my gratitude to my first mentor, the late Dr. Eugenio Prodanov. I would also like to thank Dr. Laura Castro from whom I learned a great deal about biochemical techniques and experimental design, Dr.
Beatriz Álvarez who helped me with all the hard chemistry and enzymology, and Dr
Gerardo Ferrer-Sueta from whom I learned so much, from kinetics to grammar! Beyond scientific knowledge, Dr Gerardo Ferrer-Sueta, Dr. Leonor Thomson, Dr Ana Denicola and Dr. Noriko Hikichi offered me friendship, encouragement and showed me the kind of scientist I wanted to be. I hope I will get there eventually!
During the past six years living away from friends and family I have been lucky enough to have great friends around who have been my family away from home and without whose support I would not have been able to finish. I would especially like to thank Pasquale Fulvio, Debra Prvanovic, Carolina Vázquez, Roberto Hodara, Sami
Chaouki and Tremie Gregory. Tremie you have been the voice of reason every time my
xvii
Latin passion got in the way. You have been the strength I needed when I felt I did not have enough, and the comfort I needed when things did not look very bright.
There are no words to express how much I thank my family. Leo Martín and Suky
Medina-Day, you know you are my siblings even though we have different parents. The love, friendship and support you have offered me throught the years are priceless. My love and thanks go to my sister Risel who has always been my role model. Ri, I have always looked up to you and you are the best sister anybody could have. Thank you for teaching me that book smart is not enough! Last but not least by any means, my gratitude goes to mom and dad. Your unconditional love and encouragement have been the driving force to pursue my goals. You have always trusted my decisions and always supported my dreams. You gave me all the tools you thought I could need to succeed in life and trusted me to use them correctly. I love you dearly and will always be thankful for everything you have given me. (Por último pero no por ello menos importante mi gratitud para mamá y papá. Su amor y aliento incondicional han sido el motor para perseguir mis objetivos. Siempre han confiado en mis decisions y siempre me han apoyado en mis sueños. Ustedes me dieron todas las herramientas que pensaron yo podría necesitar para tener éxito en la vida y confiaron en que yo las usara correctamente. Los amo muchísimo y siempre voy a estar agradecido por todo lo que han hecho por mi).
Edward Suarez Moreira
January 2010, Kent, Ohio
xviii xix
Abstract
Cobalamins (vitamin B12 derivatives) are essential cofactors for two enzymes in
mammals: cytosolic methionine synthase and mitochondrial methylmalonyl-CoA mutase.
In addition to its function as cofactor, a further role as a modulator of inflammatory and
immune processes has been suggested for vitamin B12. Vitamin B12 deficiency is a major
health problem in the US population, especially amongst the elderly. The vascular
endothelium lacks the transsulfuration pathway and hence, relies solely on the B12- dependent methionine synthase to metabolize homocysteine. Thus, vitamin B12 deficiency is the primary modifiable cause of hyperhomocysteinemia, a risk factor for cardiovascular disease, in the post folate fortification era. Although vitamin B12 has been
studied for over half a century, there remains much to be discovered regarding the
biochemical pathways that lead to the synthesis of the cobalamin cofactors and the
potential roles of vitamin B12 beyond its function as cofactors of methionine synthase and
methylmalonyl-CoA mutase. This research project has been designed to shed light on the
key intermediates required for the metabolism of B12 in human cardiovascular cells and
the effects of B12 status on vascular pathophysiology, independent of its actions as an
enzymatic cofactor.
xix
Resúmen
Las cobalaminas (derivados de vitamina B12) se necesitan como cofactores de dos
enzimas en mamíferos: la enzima citosólica metionina sintasa y la enzima mitocondrial, metilmalonil-CoA mutasa. Además de la función de la vitamina B12 como cofactor, se
especula que tiene un rol como modulador de los procesos inflamatorios y la respuesta
inmune. La deficiencia de vitamina B12 es un problema de salud importante en la
población de los EEUU, especialmente entre los adultos mayores. El endotelio vascular
no posee las enzimas necesarias para tener una vía de transulfuración funcional y por lo
tanto sólo puede metabolizar homocisteína por medio de la metionina sintasa dependiente
de vitamina B12. En esta era, en la cual las harinas se fortifican con folato, la deficiencia
de vitamina B12 es la causa primaria de hiperhomocisteinemia, que es un factor de riesgo
para enfermedades cardiovasculares. A pesar de que la vitamina B12 se ha estudiado por
más de medio siglo, hay mucho que descubrir acerca de los efectos que las cobalaminas
tienen mas allá de sus funciones como cofactores de la metionina sintasa y la
metilmalonil-CoA mutasa y con respecto a las vías metabólicas necesarias para la síntesis
de estos cofactores. Este proyecto de investigación se diseñó para dilucidar los
intermediarios clave necesarios para el metabolismo de B12 en células cardiovasculares
humanas y esclarecer el efecto de la vitamina B12 en la fisiopatología vascular
independiente de su función como cofactor enzimático.
xx 1
CHAPTER 1
Introduction
1.1 Cobalamin structure and catalytic function
In 1934 George Whipple, George Minot, and William Murphy shared a Nobel prize
“for their discoveries concerning liver therapy in cases of anemia” (1). The experiments conducted initially by Whipple on anemic dogs and expanded to humans suffering from pernicious anemia by Minot and Murphy, led to the discovery of “the anti-pernicious anemia” factor present in raw liver (1). Pernicious anemia is a megaloblastic anemia that presents with glossitis, neuropathic pain and paresthesias (2). Whipple demonstrated that liver, kidney and other meats contained a factor that stimulated hemoglobin production in bone marrow and suggested that patients with pernicious anemia might lack this factor.
Further studies implicated a dietary substance being involved (3). In 1926 Minot and
Murphy provided further weight to this hypothesis by reporting the successful treatment of 45 patients suffering from pernicious anemia by tube-feeding them with liver (4). The unknown compound, now known as vitamin B12, remained elusive for over 20 years until
two independent labs reported its isolation and characterization in 1948 (5-9). However,
the chemical nature of vitamin B12 was not unraveled until Dorothy Hodgkin resolved its
crystal structure in 1956 (10), research that garnered her the Nobel prize in 1964 (11). It
is now known that pernicious anemia is caused by a defect in gastric intrinsic factor, a
protein secreted by parietal cells of the stomach, resulting in impaired B12 absorption (2).
1 2
1.1.1 Cobalamin Structure
Vitamin B12 derivatives or cobalamins (Cbls) are macrocyclic compounds which
are amongst the most complex cofactors in nature. They belong to the corrinoid family,
with a macrocycle corrin ring consisting of four partially reduced pyrrole rings (pyrroline
rings), three joined at the α position through a methine linker and two by a direct bond
between the α-C of the rings (Figure 1.1) (12). The corrin is structurally similar to other
tetrapyrrolic macrocycles including the heme porphyrins of hemoglobin and
cytochromes, and the chlorins of chlorophylls. Since the corrin is more reduced than the
porphyrins and chlorins, they are thought to be more primitive molecules (Figure 1.1).
Their less conjugated character allows for certain flexibility and deviation from the corrin
plane which folds upward about the C10 - Co axis (Figure 1.2) (13). This feature is
important for the catalytic mechanism of B12-dependent enzymes (14).
Cbls or vitamin B12 derivatives (Figure 1.2) have identical substituents on the
corrin ring and a Co atom tethered in the center of the corrin plane by the four pyrrolic
nitrogens. Corrinoids incorporate a range of nucleotides. In Cbls the fifth coordination
site, called the α-axial (or lower) site, is occupied by a 5,6-dimethylbenzimidazole
(DMB) ligand, which is linked to the C17 of the D ring via an α-D-ribofuranose
phosphate (Figure 1.2). The sixth axial coordination site, or the β-axial (or upper) site,
can be occupied by a variety of ligands when the cobalt is in the +3 oxidation state. The
Cbl is then named according to the nature of the β-axial ligand (12). In vitamin B12, the
β−axial coordination position is occupied by cyanide (CN-), and the Cbl is called
- cyanocobalamin (CNCbl). When the β-axial position is occupied by OH , H2O, methyl
3
corrin chlorin porphyrin
A B A B A B NH N N HN N HN
NN NH N NH N DCC DCD
Figure 1.1. Structure of the three tetrapyrrolic macrocycles. Rings A and D are directly bound in the corrin ring, whereas they are bridged by a methine in chlorins and porphyrins. The corrin is less conjugated than its tetrapyrrolic relatives, which leads to greater conformational flexibility.
4
H2NOC CONH2
CH3 H2NOC CH3
H C 3 CONH2 N X N H3C Co C10 H N N
CH3
CH3
H2NOC H3C CH3 B4
CONH2 HN O CH N 3 H3C B2 H dimethylbenzimidazole HO N (DMB) CH3
O O B7 P O R1
O O– OH
Figure 1.2 Structures of naturally occurring cobalamins: X denotes the β-axial ligand. X = 5'-deoxyadenosyl, adenosylcobalamin (coenzyme B12); X = CH3,
2- methylcobalamin; X = H2O/OH, aquacobalamin/hydroxycobalamin; X = SO3 ,
- - sulfitocobalamin; X = NO2 , nitrocobalamin; X = CN , cyanocobalamin; X = glutathione,
glutathionylcobalamin. The positions of the protons that resonate in the aromatic region in the 1H NMR spectrum of cobalamins (B2, B4, B7, R1 and C10) are also shown. 5
(CH3), or 5’-deoxy-5’-adenosyl, the corresponding cobalamins are named
+ hydroxycobalamin (HOCbl) or vitamin B12a, aquacobalamin (H2OCbl ) or vitamin B12b, methylcobalamin (MeCbl), and adenosylcobalamin (AdoCbl) or coenzyme B12, respectively (Figure 1.2). The crystal structure of AdoCbl revealed a unique Co - C bond, the first organometallic bond described in a naturally-occurring compound. The nature of this bond is paramount in the catalytic activity of B12-dependent enzymes. The structure
of the second organometallic cofactor, MeCbl, was not resolved until 1985 (15).
The oxidation state of the cobalt in Cbls is +1 in cob(I)alamin (Cbl(I)) or vitamin
B12s, , +2 in cob(II)alamin (Cbl(II)) or vitamin B12r, and +3 in cob(III)alamins (Cbl(III)),
respectively (12). In the +3 state the cobalt coordination environment is pseudo-
octahedral whereas the +2 and +1 states are penta- and tetra-coordinate, respectively (13).
Therefore, electron transfer reactions involving Cbls are accompanied by changes in the
number of axial ligands and hence, depend upon the nature of these ligands. α-Axial
coordination of the DMB moiety and a strong σ-donating ligand at the β-axial site
stabilize the Co in its +3 state (16). The biological activity of the Cbl cofactors is
primarily associated with the cleavage of the organometallic bond. Co - C homolytic
bond cleavage yields an alkyl radical and involves a formal one electron (e-) reduction of
the Co yielding Cbl(II). Cbl(II) is a radical species and hence, reacts rapidly with alkyl
and non-alkyl radicals. The Co – C bond of Cbls can also be heterolytically cleaved in a
formal 2 e- reduction of the Co yielding Cbl(I). In this process both axial bonds are cleaved since Cbl(I) is tetra-coordinate (16).
5 6
1.1.2 Catalytic Function
Herbert Weissbach, Bob Smythe and Horace Barker carried out detailed studies on the conversion of glutamate to β-methylaspartate in the glutamate metabolism of
Clostridium tetanomorphum in the 1950s. They isolated the cofactor required for the 1,2 isomerization involved in the glutamate conversion to aspartate and identified it as a vitamin B12 derivative which contained adenine (17). Subsequently the actual structure of
this coenzyme was elucidated by the Hodgkin laboratory and shown to contain a 5’-
deoxyadenosyl group linked to the Co β-axial site via the C5 (18). By that time vitamin
B12 had been implicated in methionine biosynthesis and in 1962 it was proposed that
MeCbl is an essential component of the catalytic mechanism of methionine synthase
(MS) (19). Today three types of B12 dependent enzymes are recognized:
methyltransferases, isomerases and reductive dehalogenases (20).
Dehalogenases
The energy metabolism of various bacteria involves the dechlorination of
aliphatic and aromatic hydrocarbons. A number of reductive dehalogenases have been
identified and all of them except one have been found to have a bound corrinoid and two
Fe/S clusters. Vitamin B12-dependent dehalogenases are exclusive to prokaryotes (20).
Dehalogenating bacteria are important in the detoxification of aromatic and
aliphatic chlorinated persistent organic pollutants, such as chlorinated phenols,
chlorinated ethenes, and polychlorinated biphenyls. Different species have different
preferences for the chlorine substituent they can remove. Nevertheless they all catalyze
the following general reaction:
7
RCl + 2e- + 2H+ RH + HCl (1.1)
The Cbl-dependent reductive dehalogenases are localized to the membrane or
attached to it by a small anchoring protein. This specific location is important in linking
the dechlorination reaction to the electron transport pathway of anaerobic respiration.
Methyltransferases
Heterolytic Co – C bond cleavage plays a key role in the activity of Cbl-
dependent methyltransferases. The nucleophilic demethylation of MeCbl yields Cbl(I), a
supernucleophile that can be readily methylated using a variety of methyl donors. In
bacteria, anaerobic acetogenesis, methanogenesis and catabolism of acetic acid to
methane and CO2 depend on enzymes that use B12 derivatives for methyl transfer reactions (20).
Methionine Synthase (EC 2.1.1.13): The enzyme methionine synthase (MS) catalyzes the methyl transfer from N5-methyltetrahydrofolate (MeTHF) to homocysteine
(Hcy) to yield methionine (Met) and tetrahydrofolate (THF). In bacteria this enzyme catalyzes the final step of the de novo synthesis of Met. However in humans, Met is an essential amino acid and the function of MS is to metabolize the toxic amino acid Hcy and to generate THF which is needed for purine, pyrimidine and amino acid biosynthesis.
Thus MeCbl-dependent MS regulates S-adenosylmethionine (SAM)-dependent
methylation reactions. In the human brain all methylation reactions depend exclusively
on Cbl-dependent MS. The enzyme contains a consensus B12-binding motif GXXHXD.
The crystal structure of the Cbl binding domain of MS shows that the Cbl cofactor is in the base-off conformation with the nitrogen atom (N) of the DMB moiety situated over
8
14 Å away from the Co. The α-axial site is instead occupied by a histidine residue of MS in the so-called base-off/His-on conformation (20). Binding of MeCbl to MS catalyzes successive transmethylations in which a methyl group is transferred from MeTHF to
Cbl(I) to form THF and MeCbl. Then Hcy is methylated by accepting the methyl group from MeCbl. The proposed reaction scheme is depicted in Figure 1.3. The methyl group in MeTHF is bound to the N of a tertiary amine. A positive charge on this N makes it a much better methyl donor so it has been hypothesized that the tertiary amine accepts a H+ from an amino acid residue of MS (21). The quaternary N is a much better methyl donor and the accepting Cbl(I) is a strong nucleophile and a poor base, so the transfer of the methyl group is favored over the possible protonation of the Co(I). The second half of the reaction involves the transfer of the methyl group to the thiolate of Hcy. It has also been suggested that the essential Zn atom present in MS acts as a Lewis acid stabilizing the thiolate form of Hcy, thus favoring the nucleophilic attack to MeCbl. During the catalytic turnover the alkylation of Cbl(I) by MeTHF competes with the oxidation of the metal center. The oxidation of Cbl(I) renders the enzyme inactive. However, the enzyme can be reactivated by NADPH-dependent reduction and realkylation for which SAM is the methyl donor (22).
1,2-Isomerases
AdoCbl-dependent enzymes catalyze 1,2-intramolecular rearrangements. This group of enzymes can be subdivided into three classes: a) mutases which catalyze a 1,2 rearrangement of the carbon skeleton; b) eliminases which catalyze a 1,2 migration and
9
Homocysteine Methionine NH2 O
OH S HS HO CH3 O NH2
(1)
CH3 Co(I) Co(III) N N MeCbl (2)
H4Folate CH3H4Folate
Figure 1.3. Reaction scheme for the MeCbl-dependent methionine synthase methylation of Hcy. (1) The methyl group from MeCbl is transferred to the thiol of Hcy to yield Met and Cbl(I). (2) Cbl(I) then attacks MeTHF to generate THF and MeCbl. 10
subsequent elimination of a hydroxyl group and c) aminomutases which catalyze the migration of an amino group to an adjacent carbon.
Methylmalonyl-CoA mutase (EC 5.4.99.2): In mammals methylmalonyl-CoA mutase (MMM) is a mitochondrial matrix enzyme that catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA in the catabolism of branched amino acids. The enzyme binds the cofactor in a base-off/His-on conformation and features the same
GXXHXD B12-binding motif as found in MS. The overall reaction is depicted in Figure
1.4. The first step involves the homolytic cleavage of the Co - C β-axial bond of AdoCbl
to generate Cbl(II) and a carbon-centered deoxyadenosyl radical. The enzyme accelerates
this homolysis by a factor of 1012. The second step is the abstraction of a H atom from the
methylic C of methylmalonyl-CoA by the deoxyadenosyl radical. The primary radical generated in the substrate rearranges to a secondary radical centered on the adjacent α−C with the concomitant migration of the CoA thioester. Finally the secondary radical abstracts a H back from 5’-deoxyadenosine to generate succinyl-CoA and the original
5’-deoxyadenosyl radical that then recombines with Cbl(II) to generate AdoCbl. This 1,2-
isomerization does not involve exchange of H with the solvent (20).
1.2 Cobalamin metabolism in mammals
1.2.1 Transport and Processing
Cbl is absorbed in the small intestine bound to intrinsic factor (IF) and appears to
be transferred to transcobalamin (TC) within enterocytes (23). Cbl circulates in plasma
-14 -10 -12 bound to haptocorrin (HC; KD ~ 1x10 M)(24) and TC (KD=10 –10 M)(24, 25). In
10 11
Figure 1.4. Reaction scheme for the isomerization catalyzed by methylmalonyl-CoA mutase. Conversion of methylmalonyl-CoA to succinyl-CoA. In step (1) homolytic cleavage of the Co-C bond results in formation of a carbon-centered adenosyl radical
(Ado•) and Cbl(II). Ado• abstracts a hydrogen (H) from methylmalonyl-CoA generating a
carbon-centered methylmalonyl-CoA radical (2). The thioester group migrates to the
adjacent C, leaving a C-centered radical where the thioester was bound (3). In step (4), the product radical re-abstracts the same H from AdoH to give the product succinyl-CoA and Ado•.
12 14 2 N Co(II) Ado CH H SCoA (3) SCoA O OH OH O O O (4) 2 N Co(II) Ado CH H H SCoA A o C S OH O OH Succinyl-CoA O O O (2) SCoA 2 N Co(II) Ado CH O OH H Methylmalonyl-CoA Methylmalonyl-CoA O (1) 2 2 N Ado CH Co(III) N Co(II) Ado CH AdoCbl
13
vivo, 20% of the circulating TC is complexed with Cbl (holoTC). TC plays a critical role in Cbl transport. HoloTC is recognized by a specific transcobalamin receptor (TCR), which has been recently characterized and cloned by Quadros et al. (26). The complex then enters the cell by TCR-mediated endocytosis (26). The TC serum concentration varies between 30-120 pM for holo-TC and 300-600 pM for Cbl-free TC (apo-TC). Upon entering the cell, a series of events need to occur before Cbl can be converted to MeCbl or AdoCbl, cofactors for MS and MMM, respectively. These events are believed to include: a) dissociation of TC-Cbl from TCR in acidic endosomes; b) transport of Cbl out of the lysosomes; c) removal of the β-ligand; d) transport of Cbl to the mitochondria for
AdoCbl biosynthesis and e) cytosolic biosynthesis of MeCbl.
Much of what is known about the metabolism of vitamin B12 comes from studies
on patients with inborn errors in Cbl metabolism. Initially, the molecular bases for these
disorders were unknown and thus were classified by letter or cobalamin mutant class,
cobalamin A through H (cblA-H) and mut, based on complementation studies with
patient skin fibroblasts (27, 28). The exact biochemical pathway(s) that lead to Cbl
cofactor biosynthesis and delivery to the B12-dependent enzymes have not yet been
completely elucidated. However, many of the proteins responsible for a particular
pathologic phenotype have been identified. Figure 1.5 depicts the current understanding
of Cbl metabolism. The cblF mutation is associated with an impaired ability to export Cbl
out of the lysosome resulting in accumulation of B12 in these vesicles (29). The β-ligand
transferase responsible for the initial removal of the β-ligand allowing for further
processing of B12, is affected by the cblC type mutations (30). The CblD type mutations
13 14
Figure 1.5. Diagram of Cbl metabolism. The pathways leading to incorporation of cofactors into the B12-dependent enzymes are shown. HoloTC is recognized by the TCR and internalized via receptor-mediated endocytosis. The CblF protein is involved in the export of Cbl from the lysosome. The MMACHC protein coded by the cblC locus is an enzyme involved in the removal of the β-axial ligand. The cblD product named
MMADHC is involved in Cbl transport to the mitochondria. The cblB locus encodes the
enzyme responsible for the synthesis of AdoCbl. The cblA locus encodes for a GTPase
whose function appears to protect MMM from oxidative inactivation, and mut is the
locus corresponding to MMM. cblG encodes for MS and the cblE locus encodes for an
accessory reductase.
15
TC-Cbl
TCR
TC-Cbl Lysosome cblF Cbl cblB cblD cblC 2+ cblD AdoCbl Cbl Cbl2+ cblA cblE MCoA SCoA cblG mut MeCbl
Met cblG Hcy
16
affect the MMADHC (methylmalonic aciduria, cblD type, and homocystinuria) gene. The
MMADHC protein is essential for the biosynthesis of both cofactors and has a mitochondrial targeting sequence (31). The cblC defect has been attributed to mutations in the MMACHC gene, which encodes for a cytosolic Cbl chaperone protein called methylmalonic aciduria and homocysteinuria type C protein (MMACHC), needed for cofactor biosynthesis. It was recently shown that the MMACHC protein catalyzes the reductive decyanation of CNCbl in vitro and in vivo and the dealkylation of alkylCbls in vivo (32, 33). In this study it was shown that MMACHC did not require GSH for its catalytic function as a decyanase. However in another paper published shortly afterwards by the same groups, Kim et al. showed that GSH is required for the MMACHC-mediated dealkylation of Cbls (30). These studies finally identified the elusive decyanase or β- ligand transferase, whose role in Cbl metabolism is depicted in Figure 1.5. The sequence of the MMACHC protein was reported by Lerner-Ellis et al. (34). The protein has two motifs that are similar to the sequences in bacterial Cbl-related genes. The Cbl-binding motif is 52% identical to the corresponding motif of MMM of Streptomyces avermitilis
(34). The loci affecting mitochondrial B12 metabolism are that of MMM (mut), a GTPase
of unknown function (cblA) and Cbl adenosyltransferase (cblB), which not only catalyzes
the synthesis of AdoCbl from Cbl(I) and ATP, but also acts as a chaperone to deliver the
cofactor to MMM (35-38). The cblG and E type of mutations affect only the cytosolic
metabolism of B12. Studies by Gulati et al. (1997) showed that cblG may be attributed to
mutations in the MS gene and cblE is associated with defects in reductases required for
MS activation (22, 39).
16 17
Thiols in Cobalamin Processing
There are several chemical arguments that support thiol-dependent Cbl metabolism. It is known that thiolatocobalamins can be reduced by free thiols, yielding
Cbl(I), which, in turn, can be methylated by SAM to form MeCbl (40, 41). Moreover,
MS is reported to exhibit an intrinsic thiol oxidase activity, which suggests that thiols are involved in the reduction of Cbl (22). It has also been shown that thiols can reactivate inactive MS (22), which is in agreement with thiol-dependent synthesis of MeCbl. Due to the supernucleophilic nature of Cbl(I), an intermediate in MS catalytic cycle, Cbl(I) can be readily oxidized, rendering an inactive MS. Finally, it has also been shown that patients with cognitive disorders respond better to combined thiol/B12 supplements
compared with B12 alone, further suggesting a potential role for thiol intermediates in B12 metabolism (42). Thus, exploring the role of glutathione (γ-glutamylcysteinylglycine,
GSH) in Cbl processing is of particular interest.
GSH is a thiol-containing tripeptide essential for many key metabolic processes, involved in amino acid transport, drug excretion, and protein folding. It is the most important low-molecular weight intracellular antioxidant (43). The reaction between
+ GSH and H2OCbl to yield glutathionylcobalamin (GSCbl) is fast and
thermodynamically favorable (44). However, whether GSH is necessary for intracellular
processing of vitamin B12 is unclear. Evidence supporting GSH involvement in Cbl
processing includes the recent discovery of GSH-dependent MMACHC-catalyzed Cbl
dealkylation (30). Furthermore, the Jacobsen laboratory was the first to show that GSCbl 17 18
is present in mammalian cells and proposed that GSCbl is a key intermediate in the biosynthesis of Cbl cofactors (45-49). However, only a few studies report the isolation of
GSCbl from biological samples, and its presence in cellular samples has been suggested to be an artifactual product of the Cbl extraction procedure. We recently offered unequivocal proof of its natural intracellular occurrence (50).
1.2.2 Cobalamin Deficiency and its Diagnosis
Mammalian brain depends exclusively on B12-dependent MS activity as the sole
Hcy methylating system (51, 52). Moreover, B12 deficiency has been identified as a risk
factor for cognitive disorders such as senile dementia and Alzheimer's disease (53). Cbl
deficiency is a serious public health problem, especially amongst the elderly and it has been estimated to affect between 10 – 40% of the elderly population (54). Cbls are
present in animal-source foods only and therefore, supplementation is paramount for
vegans and others deficient in these foods. There is no scientific evidence that corrinoids
present in spirulina, nori, kelp, chlorella and algae are biologically active, nor that foods prepared with bacterial fermentation can provide useful amounts of bioactive Cbls (55).
The most common cause of vitamin B12 deficiency is inadequate intake, including
veganism, lacto-ovo vegetarianism and overall low animal-source food intake.
Malabsorption due to ileal disease, chronic pancreatitis, pernicious anemia, and chronic
gastritis and gastric atrophy can contribute to vitamin B12 deficiency. Some medications
may also interfere with Cbl absorption (55, 56).
Inborn errors of Cbl metabolism also lead to a functional B12 deficiency despite
adequate intake and absorption, as described in the previous section. The clinical
19
hallmarks of Cbl deficiency are megaloblastic anemia and neurological disease.
Megaloblastic anemia is characterized by large, immature blood cells due to inadequate
DNA synthesis which results in defective cell division. Anemias related to Cbl deficiency are easy to diagnose, and when treated in a timely fashion, completely curable. The problem lies in diagnosing patients with subtle Cbl deficiency without overt symptoms of anemia. If treatment is delayed, the neurologic damage can be irreversible (56).
Even though Cbl deficiency is an important public health problem, there is no gold standard to diagnose Cbl deficiency. Total plasma Cbl has been used as an indicator of Cbl status since the 1950s. It is easily accessible and a relatively inexpensive assay.
Patients with levels below the reference interval are considered B12 deficient. However,
the normal range is debatable since there is great variation in the confidence interval (55).
Moreover, altered levels of HC can lead to false positives and false negatives (56).
Normal plasma B12 concentrations are greater than 250 pM, and patients with levels
below 125 pM are considered B12 deficient (55). Intermediate values call for functional
assessment of B12 activity. Impairment of the catalytic function of the Cbl-dependent
enzymes results in the accumulation of Hcy and methylmalonic acid (MMA) in plasma.
Therefore, the concentrations of these two metabolites are typically used to diagnose
functional B12 deficiency (56). However, measurement of these biomarkers is not without
pitfalls. Reduced renal function increases plasma levels of MMA and Hcy leading to false
positive results (56). Additionally, plasma Hcy is affected by life style factors, folate
status and vitamin B6 status independent of Cbl status. Moreover, it has been reported
that normal levels of plasma Cbl, MMA and Hcy were detected in patients showing
20
clinical signs of B12 deficiency that reverted upon Cbl therapy (57). These shortcomings
have lead to an intensive search for a new more specific biomarker. As discussed earlier,
cells take up Cbl bound to TC via the TCR, therefore holoTC is theoretically a useful
measure of available Cbl. Nexø et al. have recently developed specific methods for
measuring holoTC and have proposed to study of the use and clinical performance of
holoTC measurements (58).
1.3 Cobalamin and Reactive Species
1.3.1 Reactive Oxygen and Nitrogen Species
Aerobic metabolism represents an energetic advantage over anaerobic
fermentation. However, fuel is not the only target for oxidation. Living in an oxygen-rich
atmosphere entails dealing with the by-products of oxygen metabolism. Mitochondrial
respiration has evolved a very efficient system to totally reduce O2 to H2O in one single 4
e- transfer process. Nevertheless it is impossible to escape the formation of partially
reduced highly reactive metabolites. Mitochondria in particular are an important source
of these partially reduced oxygen metabolites, collectively called reactive oxygen species
(ROS) (59). Aerobic organisms have evolved antioxidant defense mechanisms to protect
themselves from these potentially toxic ROS (Figure 1.6), and have also evolved
pathways that harness the chemical properties of ROS for cell signaling and defenses
against pathogens. The discovery of nitric oxide (•NO), a N-centered free radical, led to
the coining of the term reactive nitrogen species (RNS) for the species derived from •NO
21
cytochrome oxidase citocromo oxidasa
aminoaminoácido acid oxidase oxidasa
xanthinexantino oxidasaoxidase
NADPH oxidasaoxidase
-0.33 V +0.94 V +0.38 V +2.33 V .- . O2 O2 H2O2 OH H2O
α-tocoferol α-tocopherol ascorbato SOD ascorbate urato urate catalasecatalasa
2GSH GSSG
glutathioneglutatión peroxidasa peroxidase
Figure 1.6 Production and detoxification of reactive oxygen species. ROS are
depicted in 1 e- reduction steps together with their enzymatic and non-enzymatic defense
- •- systems. The redox potential for each 1 e reduction is also shown. O2 = oxygen; O2 =
• superoxide; H2O2 = hydrogen peroxide; OH = hydroxyl radical; H2O = water.
22
metabolism. It is now clear that ROS and RNS are both friends and foes that the cell must keep tightly regulated between production and destruction in order to perform normal cellular functions (58). If this balance is compromised and the cellular defenses are overwhelmed by ROS and/or RNS production, then oxidative stress ensues.
•- - •- Superoxide: Superoxide (O2 ) is formed upon a 1 e reduction of O2. O2 is a free radical, possessing 1 unpaired e-. This species is a by-product of normal cellular
metabolism, it is produced by mitochondrial and reticular membrane electron transport
systems, or enzymes such as NADPH oxidase (NOX) and xanthine oxidase (XO), and
•- uncoupled nitric oxide synthase (NOS) (60). O2 is an important ROS, levels of which
•- are augmented in acute or chronic inflammation (61, 62). O2 can damage lipids, nucleic acids and proteins but also acts as a signaling molecule by modulating the cellular response to various stressors (63). It is detoxified by enzymes called superoxide dismutases (SOD) which catalyze its dismutation to O2 and H2O2 with a second-order
rate constant of 2 x 109 M-1s-1 (64).
- Hydrogen Peroxide: Hydrogen peroxide (H2O2) is the 2e reduction product of
•- oxygen, generated by oxidases and by O2 dismutation. It is detoxified mainly by catalases and glutathione peroxidase. Its moderate reactivity and lack of charge allows it
to diffuse across the cell membranes to function as a signaling molecule (65). Lower
concentrations of this species are essential for maintenance of physiological function and signaling. H2O2 is involved in the control of coronary blood flow (66), angiogenesis (67,
68), wound healing (69) and regulation of cell survival (70). However, it readily oxidizes metal centers generating highly reactive, strong oxidants such as •OH and oxoferryl metal
22 23
centers (FeIV=O) (71). Higher concentrations are associated with pro-apoptotic and pro-
inflammatory actions (59).
Nitric Oxide: •NO is a small nitrogen-centered free radical that can diffuse several
cell diameters from its production site. It is synthesized by the family of enzymes called
nitric oxide synthases (NOS). It serves as a neurotransmitter involved in memory and
learning, and as a signaling molecule controlling vascular tone, cell proliferation, and cell
survival (72). During inflammation an inducible form of NOS, (iNOS) produces large
quantities of •NO involved in host protection against pathogens. However, when produced in large quantities it has cytotoxic effects that could be mostly ascribed to the production of strong oxidant peroxynitrite (ONOO-) (72).
•- • - Peroxynitrite: O2 and NO react at a diffusion controlled rate to yield ONOO .
This is a strong oxidant capable of directly or indirectly oxidizing metal centers, thiols, and nitrating aromatics (73). ONOO- also gives rise to other strong oxidants such as
•- • • carbonate radical (CO3 ), nitrogen dioxide ( NO2), and hydroxyl radical ( OH). Its
overproduction has been implicated in the pathogenesis of several inflammatory diseases,
including arteriosclerosis (73).
1.3.2 Cobalamin and Reactive Species
The intermediates in the catalytic cycle of both MS and MMM are readily
oxidized by ROS and RNS rendering the enzymes inactive. Low MS activity will lead to
accumulation of Hcy, which in turn has been established to induce oxidative stress,
further impairing MS activity thus perpetuating a vicious oxidative cycle.
24
Cbl(II) reacts with •NO to form nitroxycobalamin (NOCbl) and Cbls are capable of inhibiting some •NO-mediated processes. Cbl reverses •NO-dependent MS inhibition in a
model of •NO induced neural tube closure defects (74) and •NO-mediated inhibition of
cell proliferation (75), and inhibits •NO-mediated vasodilation (76). However, there are
numerous misleading reports in the biochemical literature that HOCbl reacts directly with
•NO to form NOCbl (75, 77-80). Even though there has been much debate in the
+ literature regarding this reaction (81-84), it is now accepted that H2OCbl /HOCbl
(oxidized state; Cbl(III)) does not react directly with •NO to yield NOCbl (83, 84).
Furthermore, the reaction observed by some authors when studying the direct interaction
of HOCbl with •NO is not due to the formation of the extremely air-sensitive, well
characterized NOCbl (λmax = 318, 350 (shoulder) and 479 nm)(81), but instead from the
conversion of HOCbl to air-stable nitrocobalamin, NO2Cbl (λmax = 354, 413 and 523 nm)
• (85). That is, trace amounts of O2 from air oxidizes NO to nitrite, which then binds rapidly to HOCbl (K = 2.2 x 105 M-1, k = 99.8 M-1 s-1, at 25 °C) (86, 87). Importantly,
cells have the ability to reduce cob(III)alamins (including HOCbl) to cob(II)alamin
(Cbl(II)) (33), and it is Cbl(II), not HOCbl, which has the ability to scavenge •NO, at
rates approaching the rate of diffusion to form NOCbl (K ~ 1 x 108 M-1, k = 7.4 x 108 M-1 s-1 25 °C) (84, 88). This might account for the inhibition of NO-driven processes
previously attributed to HOCbl.
On the other hand Cbl has been reported to exhibit antioxidant effects in Hcy-
independent systems (89-91). The possible mechanisms to explain these antioxidant
effects are addressed in this dissertation.
25
1.4 Cobalamin and Vascular Biology
1.4.1 The Vascular Endothelium
The vascular endothelium is the cell monolayer that lines the inner wall of blood vessels. Far from being an inert barrier between the blood and the vessel, the endothelium plays a key role in vascular control and homeostasis. The endothelium participates in the regulation of vascular tone, nutrient delivery, waste removal, inflammation, thrombosis and coagulation (92). The secretion of autocrine and paracrine mediators by the endothelium accounts for many of its regulatory functions. The endothelium secretes
•NO, prostaglandins, and angiotensin II among other molecules. Pro-inflammatory
stimuli such as TNF-α and IL-1 activate the endothelium leading to the expression of
adhesion molecules and chemokines. The activated endothelium is also responsible for recruitment, adhesion and diapedesis of leukocytes into the vascular wall via the secretion of chemokines such as MCP-1 and IL-8 (93) and the expression of selectins and integrins (94). Individuals with cardiovascular disease (CVD) or with risk factors for
CVD have impaired endothelial function (95, 96). These observations lead to the conclusion that endothelial dysfunction is an important component in the pathophysiology of CVD (92)
Maintaining adequate levels of •NO is a key role of the endothelium, achieved by
the constitutively expressed endothelial NOS. •NO induces vasodilation via the activation
of guanylate cyclase in the smooth muscle and inhibits smooth muscle proliferation, leukocyte adhesion, and platelet aggregation (97). Reduction in •NO bioavailability is a
key feature of endothelial dysfunction.
26
1.4.2 Oxidative Stress and Cardiovascular Disease
Chronic and acute overproduction of ROS and RNS under pathophysiologic conditions is a hallmark in the development of cardiovascular disease (CVD). These reactive species are generated by NOX, XO, lipoxygenase, uncoupling of NOS, myeloperoxidase or the reticular and mitochondrial electron transport systems. ROS are important signaling molecules underlying vascular inflammation. Several animal models of oxidative stress support the idea that ROS have a causal role in CVD. Moreover, oxidative stress is the unifying mechanism for many CVD risk factors (98).
All the cells in the atherosclerotic blood vessel show increased production of
ROS: endothelial cells, leukocytes, fibroblasts, and smooth muscle cells. Elevated levels
•- • of O2 lead to a more efficient scavenging of NO resulting in the formation of the strong
oxidant ONOO- and in an impaired dilation response of the vessel. ONOO- formation is
evidenced by the presence of nitrated tyrosine residues in the atherosclerotic lesion. The
increased ROS production in the atherosclerotic vessel leads to LDL oxidation. Oxidized
LDL induces a series of pro-atherogenic effects such as changes in the extracellular
matrix and endothelial cell apoptosis (98).
Even though mitochondria, XO and uncoupled NOS have also been implicated in
•- the pathophysiological generation of O2 in CVD (98), it appears that vascular NOX present in fibroblasts, smooth muscle cells and endothelial cells account for the majority
•- •- of the vascular O2 (99). This species, or secondary oxidants derived from O2 , oxidize low density lipoprotein (LDL), reduce •NO availability and induce endothelial apoptosis.
Angiotensin II, platelet derived growth factor and TNF-α are agonists capable of
27
activating NOX (98). In humans, the relevance of NOX-derived ROS is evidenced by the colocalization of the p22phox subunit and oxidized LDL in atherosclerotic human coronary arteries (100).
There is evidence that oxidative stress and particularly activation of NOX is involved in the pathophysiology of heart failure (98). ROS levels were elevated in the failing myocardium of patients with ischemic or dilated cardiomyopathy (101). TNF-α and platelet NOX were elevated in patients with heart failure (102) and translocation of the regulatory subunit of NOX to the membrane fraction was observed in the failing human heart (103).
Hypertension is a major cause of stroke, atherosclerosis, peripheral vascular disease, renal disease, congestive heart failure and left ventricular dilation. Angiotensin II modulates blood pressure via the renin-angiotensin system and via stimulation of AT1 receptors in the vasculature. Stimulation of this receptor induces ROS production through activation of NOX (104).
Oxidative stress has been shown in animal models of brain ischemia where lack of SOD exacerbated neuronal injury and over-expression of SOD had a protective effect
(98). Oxidative marker levels were augmented in plasma of stroke patients (105) and phagocytes of patients with ischemic stroke showed an increase in ROS production (106).
As discussed above, oxidative stress is at the core of CVD pathophysiology. Hence, there is great interest in the development and characterization of safe antioxidants for the treatment of CVD.
28
1.4.3 Cobalamin and Hyperhomocysteinemia
Impairment of the B12-dependent MS leads to hyperhomocysteinemia, an independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular
disease (107-110). Homocysteine (Hcy) is an amino acid generated in the methionine
cycle. It can be re-methylated to methionine or be metabolized to cysteine. There is
considerable evidence that elevated plasma total Hcy can adversely affect the vascular
endothelium (111). Hcy is also a mitogen for smooth muscle cells (112), and proliferation
and migration of smooth muscle cells into the intimal space is a hallmark of
atherogenesis. It has been shown that in end-stage renal disease patients, total Hcy correlates with the onset of atherosclerosis and thrombosis (113).
B12 deficiency is a common and significant public health problem, particularly
amongst the elderly (114). Evidence suggests that B12 deficiency may contribute to the
risk of vascular disease (115, 116). Since the introduction of the folic acid fortification of
the US diet, B12 deficiency ranks well above folate deficiency as the primary modifiable
cause of hyperhomocysteinemia (110, 114, 117).
The metabolic pathways of Hcy are shown in Figure 1.7. In order to maintain low
steady-state intracellular levels of Hcy, cells re-methylate Hcy to methionine (Reactions 1
and 2), convert it to Cys through the transsulfuration pathway (Reactions 3 and 4), or
export it to circulation. The MS dependent pathway is present in all cells (Reaction 1),
and it is folate and B12 dependent. The second re-methylation pathway (Reaction 2),
catalyzed by betaine: Hcy methyltransferase (BHMT), is found in human liver and
kidney only (51, 118). The transsulfuration pathway (Reactions 3 and 4) is present in
29
Protein
ATP PPi + Pi
Methionine SAM Methyl Acceptor THF dimethylglycine
MeCbl 21 Methionine 5 Cycle CH3THF Betaine Methylated -Ado Product Homocysteine SAH +Ado +Ado 3 Transsulfuration Cystathionine Pathway
+Ado 4 GSH
2- Protein Cysteine SO4
Protein
Figure 1.7. Homocysteine metabolic pathways. Hcy levels are maintained by re- methylation (Reactions 1 and 2), conversion to Cys through the transsulfuration pathway
(Reactions 3 and 4), or export to the circulation. The MS-dependent re-methylation is present in all cells (Reaction 1), and it is folate and B12 dependent. Re-methylation can
also be catalyzed BHMT (Reaction 2), found in liver and kidney. The transsulfuration
pathway (Reaction 3 and 4) is vitamin B6 dependent. CBS (Reaction 3) is not present in
cardiovascular tissue. The methionine cycle provides the universal methyl donor SAM
which serves as a substrate for a range of methyl transferases (Reaction 5).
30
liver, kidney, pancreas and small intestine, and it is vitamin B6 dependent. However,
cardiovascular cells and tissues are not capable of converting Hcy to Cys through the
transsulfuration pathway because they do not express cystathionine β-synthase (CBS),
(119, 120). Although low levels of CBS activity have been detected in human umbilical
vein endothelial cells in culture (121), Chen et al. reported that human cardiovascular
cells did not express CBS nor BHMT (120). Human aortic endothelial cells (HAEC) rely
solely on B12-dependent MS to metabolize Hcy (122) thus, vascular Hcy homeostasis
should be particularly sensitive to B12 levels. Moreover, Quadros et al. have shown that the vascular endothelium secrets functional TC (123) and Nexø et al. have reported that holoTC is an early marker of changes in Cbl homeostasis (124), supporting an important role of the vascular endothelium in Cbl homeostasis.
1.5 Non-cofactor functions of Cbl
The biochemistry of Cbls as cofactors of MS and MMM is well characterized.
However, additional physiological and biochemical roles for Cbl have also been suggested. Accumulating evidence suggests that Cbl has regulatory roles on the immune system, inflammation, neurophysiology and gene expression that are independent of its cofactor biochemistry (125). Cbl plasma levels of 300 pM minimize micronucleus formation in peripheral blood lymphocytes and uracil incorporation into leukocyte DNA depends on plasma Cbl concentration. In vitro, MeCbl acts as a direct methyl donor for
DNA methylation; however there is no evidence that this reaction occurs in vivo. These findings point to a role of Cbl in DNA metabolism and genomic stability (125). Cbl regulates MS gene expression (126). It has been demonstrated that Cbl regulates the 30 31
activity of MS at the translational level. The MS transcript has a highly structured 5’- untranslated region. In this region there is an internal ribosome entry site which is modulated by Cbl. This modulation is hypothesized to be mediated by a Cbl-dependent protein (126). Cbl is also associated with protective effects in neurological pathologies.
High doses of Cbl have been shown to have positive effects on nerve regeneration in rats with acrylamide induced neuropathy (127), pain reduction in a rat neuropathy model
(128), muscle action potential conduction in patients with amyotrophic lateral sclerosis
(129), and improving neurological function in different pathologies (125). Vitamin B12 is
also hypothesized to contribute to regulation of inflammatory processes. Low Cbl levels
have been associated with Alzehimer’s disease and senile dementia (53, 130, 131), which
has been hypothesized to be due to increased inflammatory processes in the brain (132).
Cbl is also used for the treatment of the inflammatory diseases, rheumatoid arthritis and
osteoarthritis (133, 134) and has been reported to resolve a case of chronic erythema
nodossum, a condition generally treated with anti-inflammatory drugs (135).
Cbl therapy has also been shown to normalize levels of TNF-α and epidermal
growth factor in Cbl deficient patients (136). The effects of Cbl on the immune function
and on modulating cytokine production might account for these clinical effects. More
recently, it has been proposed that the anti-inflammatory properties attributed to Cbl may
be related to its ability to decrease oxidative stress (137-139).
1.6 Aims
The present dissertation deals with various aspects of the chemistry and vascular
biochemistry of vitamin B12 derivatives. The study of different Cbl derivatives present in
32
human tissues and cells could provide insight into Cbl processing pathways. It could also provide information on how pathophysiological conditions differentially affect Cbl metabolism. In order to assess the intracellular Cbl profile, it is necessary to synthesize pure standards for the identification of Cbls which are not commercially available. This work starts with the description of a general procedure for the synthesis of non-alkyl cobalamins and the chemical characterization of the products. The naturally occurring derivatives present in HAEC are then determined. The accurate assessment and interpretation of intracellular Cbl profiles is discussed. The effect of GSH depletion on the intracellular Cbl profile is assessed to probe for the possible role of GSH in Cbl metabolism.
The non-cofactor roles of vitamin B12 and its possible regulatory effects on
inflammatory processes are a fascinating new field in Cbl biochemistry. This dissertation
explores the antioxidant effects of Cbl in HAEC. A model of hyperhomocysteinemia is
used as a pro-oxidant insult and the protective effect of Cbl against oxidative stress is
•- studied. Finally the reaction of Cbl(II) with O2 is studied to assess the feasibility of
•- direct scavenging of O2 by Cbl(II) to account for the antioxidant properties exhibited by
Cbl.
33
CHAPTER 2
Synthesis and Characterization of Biologically Relevant Cobalamins
2.1 Introduction
Upon entering cells, cobalamin derivatives are converted to MeCbl and AdoCbl by currently ill-defined mechanisms. Many studies have been carried out over the past few decades involving the extraction and identification of cobalamins from mammalian cells, tissue and blood, in addition to other biological samples such as foods and seaweed.
+ + + AdoCbl, MeCbl and H2OCbl (H2OCbl ↔ HOCbl + H ; pKa = 7.8) (140) are the major
cobalamin metabolites isolated from biological samples (141-144). Sulfitocobalamin
- (SO3Cbl ) is also isolable from mammalian cells and foods (45, 48, 143-151), and there
are also reports on the isolation of nitrocobalamin (NO2Cbl) from biological sources
(145, 152). Whether or not cyanocobalamin is truly "naturally occurring" is controversial; some studies report small amounts of this derivative, especially in smokers (141, 153).
Thiol derivatives of B12, thiolatocobalamins (RSCbl), were first identified in the
1960s, but have not attracted much attention until recently (42, 45-49, 154-168).
Glutathionylcobalamin (GSCbl) is an important cobalamin metabolite in mammals (47-
49), and is more active than other Cbls in promoting MS activity in rabbit spleen extracts.
It has been proposed that GSCbl (or a closely related thiolatocobalamin adduct) is a
precursor of the two coenzyme forms of vitamin B12, AdoCbl and MeCbl (47). Formation
+ 8 -1 of GSCbl from H2OCbl and GSH is irreversible (Kobs ~ 3 x 10 M at pH 7.4, 25 °C)
33 34
and rapid (t½ ~ 3 s for [GSH] = 5 mM, pH 7.4, 37 °C) (44). An alternative role for
GSCbl was also recently proposed, in which the formation of GSCbl prevents B12 from
being scavenged by xenobiotics (156). Finally, McCaddon and co-workers suggested that
GSCbl and related RSCbls might be more effective than currently available
+ pharmaceutical B12 forms (CNCbl and H2OCbl ) in treating of B12-related conditions
associated with oxidative stress such as Alzheimer's disease (42, 163). However, the
+ apparent clinical benefit of combining N-acetylcysteine with H2OCbl in patients with
Alzheimers disease or mild cognitive impairment requires formal clinical trials to prove
or refute such observations (42, 169).
In the MeCbl-dependent MS reaction, a methyl group is transferred from
methyltetrahydrofolate to Hcy via MeCbl to generate Met and THF (Figure 1.4).
Impairment of this reaction leads to elevated serum levels of Hcy, which is associated
with an increased risk of cardiovascular, peripheral vascular and cerebrovascular diseases
(111, 122, 170-173). In addition, moderately raised serum Hcy levels are more prevalent in patients suffering from neurological diseases such as Alzheimer’s disease (53, 174-
176), and appear to predict cognitive decline (177, 178). The N-acetylcysteine derivative
+ of Cbl (NACCbl) is also of interest, given the benefits of co-administrating H2OCbl and
N-acetyl-L-cysteine to Alzheimer's patients (42, 169) and the possibility that NACCbl, rather than the individual components themselves, is responsible for the beneficial effects of this approach.
It was also of interest to see whether the synthetic procedure could be used to synthesize other non-thiol cobalamin derivatives, such as the sodium salt of
35
sulfitocobalamin (Na[SO3Cbl]). Although SO3Cbl is isolable from biological samples
(145-152), and is well studied and structurally characterized (86, 87, 155, 161, 166, 167,
179-187), it is not commercially available.
Finally, it is noteworthy that there are numerous studies concerning the isolation
and identification of cobalamin metabolites from biological samples which report
"unknown" analogues of Cbl (188-193). To assist in their identification, simple
procedures are required to synthesize potential candidates with different β-axial ligands,
allowing spectroscopic and chromatographic comparison of unknown cobalamin
metabolites with known standards. In addition, pure, well characterized cobalamin
derivatives will assist researchers investigating intracellular cobalamin processing.
Simple synthetic procedures are also useful when expensive, radioactive cobalamin
derivatives are desired for studies on the uptake and/or conversion of cobalamin
derivatives in humans, animals and cell models (46, 194-196).
The syntheses of four novel sulfur-containing Cbls is presented (the thiol ligand
structures are shown in Figure 2.1): D,L-homocysteinylcobalamin (HcyCbl), the sodium
salt of N-acetyl-L-cysteinylcobalamin (Na[NACCbl]), and 2-N-acetylamino-2-
carbomethoxy-ethanethiolatocobalamin (NACMECbl); and the sodium salt of
sulfitocobalamin (Na[SO3Cbl]). The kinetics of acid-catalyzed decomposition of GSCbl
and NACMECbl are also reported.
36
HO HO O O O H2N O O HS HN HN HS NH2 OH N-acetyl-L-cysteine HS O N-Acetyl-L-cysteine D,L-homocysteine HO HN O O O O
OH O HS HN glutathione HS NH2 N-Acetyl-L-cysteineN-acetyl-L-cysteine methyl methyl ester ester L-cysteine
Figure 2.1 Structures of the thiols used for thiolatocobalamin synthesis.
37
The Specific Aims of this chapter are
• To develop a general synthetic procedure to synthesize non-alkyl
cobalamins including thiolatocobalamins.
• To chemically characterize the cobalamins synthesized.
2.2 Experimental Section
2.2.1 Materials
Hydroxycobalamin hydrochloride (HOCbl•HCl), 98% (stated purity by manufacturer) was purchased from Fluka. The percentage of water in HOCbl•HCl (•nH2O)
(batch-dependent, typically 10-15%), was determined by converting HOCbl•HCl to
- -1 -1 dicyanocobalamin, (CN)2Cbl (0.10 M KCN, pH 10.0, ε368 nm = 30.4 mM cm (197)). NaN3
(99%), MES, KNO3 (99%), D,L-homocysteine was from ACROS Organics and glutathione
(GSH, 98%; i.e., in its reduced form) was purchased from Aldrich. NaOAc (99%), NaNO2
(>98%), NaSO3 (>98%) and N-acetyl-L-cysteine (≥99%) were obtained from Sigma. Water
was purified using a Barnstead Nanopure Diamond water purification system and/or HPLC
grade water. All thiol solutions were prepared directly before use.
2.2.2 General Methods
pH measurements were made with a Corning Model 445 pH meter in conjunction
with a Mettler-Toledo Inlab 423 electrode at room temperature. The electrode was filled
with 3 M KCl / saturated AgCl solution, pH 7.0. The electrodes were standardized with
standard BDH buffer solutions at pH 4.01 and 6.98. Solution pH was adjusted using HCl
or NaOH solutions as necessary.
38
1H NMR spectra were recorded on an Inova 500 MHz or a Bruker 400 MHz
spectrometer equipped with a 5 mm probe at room temperature (22 ± 1 °C). Solutions were
prepared in D2O or MES buffer (pD 5.50) and TSP (3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt) was used as an internal standard. UV/visible spectra were recorded on a
Cary 5000 spectrophotometer equipped with a thermostatted cell changer (25.0 ± 0.1 °C), operating with WinUV Bio software (version 3.00). Electrospray mass spectra were recorded using a BRUKER Esquire~LC mass spectrometer. Mass spectra for all cobalamins were recorded in the positive mode, except for sulfitocobalamin, which was recorded in the negative mode.
For experiments conducted under anaerobic conditions, solutions were degassed using three freeze-pump-thaw cycles and argon using standard Schlenk techniques. Air-free manipulations were carried out on a Schlenk line or in an MBRAUN Labmaster
130(1250/78) glovebox.
Synthesis of N-Acetyl-L-cysteine methyl ester: N-acetyl-L-cysteine methyl ester was prepared according to a modified published procedure.(198) Briefly, CH3OH (1.6 ml, 39
mmol) and concentrated HCl (180 μl) were added slowly to a solution of N-acetyl-L- cysteine (NAC, 640 mg, 3.9 mmol) in chloroform (9 ml). The reaction mixture was refluxed for 3 h at 66 °C, taken to dryness by rotary evaporation and the product recrystallized from diethyl ether. Yield: 405 mg (59%). 1H NMR: (δ, ppm) 1.29 (t CH), 2.07 (s, MeCO), 2.97 (d
CH2), 3.78 (s MeO).
Determination of the Thiol Concentrations: The total reduced thiol concentration
was determined using the Ellman’s procedure (199). One part of the thiol solution was
39
diluted with 5 parts of Ellman's reagent (32 μg ml-1 dithionitrobenzoic acid and 1.25 mM
EDTA in 0.5 M Tris, pH 8.5) and the absorbance determined at 412 nm (ε=13.6 mM-1 cm-1).
2.2.3 Synthesis of Thiolatocobalamins
All syntheses were carried out under red-light-only conditions, due to the potential light sensitivity of thiolatocobalamins (167). Unless specifically stated otherwise, all syntheses were carried out under aerobic conditions.
N-Acetyl-L-cysteinylcobalamin, sodium salt (Na[NACCbl]): A solution of N- acetyl-L-cysteine (263 μml, 284 mM, 74.7 μmol, 1.1 mol equiv.) in MES buffer (0.1 M, pH ~6) was added drop wise to a solution of HOCbl•HCl (107 mg, 67.9 μmol) in MES buffer (0.80 ml, 0.1 M, pH ~6) with stirring, and the reaction was allowed to proceed for
30 min at 0 °C. The product precipitated upon dripping into a chilled acetone solution (-
20 °C), and was filtered, washed with chilled acetone (20 ml, -20 °C) and diethyl ether
(10 ml, -20 °C). The product was dried at 50 °C under vacuum ( 2 x 10-2 mbar) overnight.
Yield: 90 mg (87%). The purity assessed by conversion to dicyanocobalamin (197) was
1 95 ± 2%. H NMR (D2O, δ, ppm): 6.10 (s C10), 6.28 (d R1), 6.40 (s B4), 6.96 (s B2),
7.20 (s B7). The cobalamin purity assessed by 1H NMR spectroscopy (200) was ~98%.
+ + ES-MS, m/z: 1492.3 (calcd for [NACCbl + 2H] , C67H98CoN14O17PS = 1492.6); 1513.9
+ + (calcd for [NACCbl + H + Na] , C67H97CoN14NaO17PS = 1514.6); 746.5 (calcd for
2+ 2+ [NACCbl + 3H] , C67H99CoN14O17PS = 746.8); 757.7 (calcd for [NACCbl + Na +
2+ 2+ 2H] , C67H98CoN14NaO17PS = 757.8).
40
2-N-Acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin (NACMECbl): The procedure was similar to that for Na[NACCbl], except that a solution of N-acetyl-L- cysteine methyl ester (NACME, 249 μml, 250 mM, 62.2 μmol, 1.2 mol equiv) in MES buffer was added to a HOCbl•HCl solution (81.48 mg, 51.8 μmol) in MES buffer, and the mixture left for 30 min to react at 0 °C. Yield: 66.3 mg (85 %). The purity assessed
1 by the dicyanocobalamin test (197) was 96 ± 2%. H NMR (D2O, δ, ppm): 6.10 (s C10),
6.30 (d R1), 6.40 (s B4), 6.95 (s B2), 7.21 (s B7). The cobalamin purity assessed by 1H
NMR spectroscopy was ~98%. ES-MS, m/z: 1506.6 (calcd for [NACMECbl + H]+,
+ + C68H100CoN14O17PS = 1506.6); 1527.9 (calcd for [NACMECbl + Na] ,
+ 2+ C68H99CoN14NaO17PS = 1528.6); 753.7 (calcd for ([NACMECbl + 2H] ,
2+ 2+ C68H101CoN14O17PS = 753.8); 765.0 (calcd for [NACMECbl + Na + H] ,
2+ C68H100CoN14NaO17PS = 764.8).
D,L-Homocysteinylcobalamin (HcyCbl): The synthesis of this derivative was
carried out under strictly anaerobic conditions. An anaerobic solution of D,L-Hcy (395
μml, 191 mM, 75.4 μmol, 1.2 mol equiv.) in MES buffer (0.1 M, pH ~6) was added drop
wise to an anaerobic solution of HOCbl•HCl (98.7 mg, 62.8 μmol) in MES buffer (0.80
ml, 0.1 M, pH ~6) with stirring, and the reaction allowed to proceed for 5 min at 0 °C.
The product precipitated upon dripping into a degassed chilled acetone solution (-20 °C),
and was filtered and washed with chilled acetone 20 ml, -20 °C). The product was dried
at 50 °C under vacuum (2 x 10-2 mbar) overnight. Yield: 64.4 mg (70%). The purity
1 assessed by conversion to dicyanocobalamin was 97%. H NMR (D2O, δ, ppm): 6.10 (s
C10), 6.28 (d R1), 6.38 (s B4), 6.95 (s B2), 7.20 (s B7). The cobalamin purity assessed by
41
1H NMR spectroscopy was ~98%. ES-MS, m/z: 1464.8 (calcd for [HcyCbl + H]+,
+ + C66H98CoN14O16PS = 1464.6); 1486.7 (calcd for [HcyCbl + Na] ,
+ 2+ [C66H97CoN14NaO16PS] = 1486.6); 732.5 (calcd for [HcyCbl + 2H] ,
2+ 2+ C66H99CoN14O16PS = 732.8); 743.4 (calcd for [HcyCbl + Na + H] ,
2+ C66H98CoN14NaO16PS = 743.8).
Glutathionylcobalamin: GSCbl was synthesized according to a published
procedure (159). A solution of GSH (605 μl, 263 mM, 159 μmol, 1.9 mol equiv.) was
added drop wise to a solution of HOCbl•HCl (129.31 mg, 82.3 μmol) in water (1.00 ml),
the vial was capped, vigorously shaken, left in the dark and allowed to react for 3 h. The
product precipitated upon dripping into a chilled acetone solution (-20 °C), and was filtered, washed with chilled acetone (20 ml, -20 °C) and diethyl ether (10 ml, -20 °C).
The product was dried at 50 °C under vacuum ( 2 x 10-2 mbar) overnight. The product
was characterized by UV/visible spectroscopy (Figure 2.2) and 1H NMR spectroscopy
(Figure 2.3), and the purity was found to be >95% as determined by 1H NMR spectroscopy.
2.2.4 Synthesis of Non-thiolatocobalamins
Sulfitocobalamin, sodium salt (Na[SO3Cbl]): A Na2SO3 solution (266 mM, 519
μl, 138.1 μmol, 1.3 mol equiv.) was added drop wise to a HOCbl•HCl solution (162.34 mg, 103.3 μmol) in MES buffer (pH 6.0, 1.60 ml), and the mixture left to react at 0 °C
(ice bath) for 2 h. The product precipitated upon dripping into acetone (20 ml, -20 °C),
and was washed with acetone (20 ml), diethyl ether (-20 °C) and dried overnight under
42
1.2
+ HAquoCbl2OCbl 1.0 NACCbl NACMECbl 0.8 HcyCbl GSCbl 0.6 Abs 0.4
0.2
0.0 300 400 500 600 700 λ (nm) Figure 2.2. UV/visible spectra of thiolatocobalamins. Electronic spectra of GSCbl,
+ NACCbl, NACMECbl, and D,L-HcyCbl, in H2O, 25 ± 1 ºC. The spectrum of H2OCbl is
also shown for comparison purposes.
43
A B
7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 6.06.26.46.66.87.07.27.4 ppm ppm
C D
6.32 6.28 6.24 6.106.15 6.05
7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 7.4 7.07.2 6.8 6.46.6 6.2 5.86.0 ppm ppm
Figure 2.3. 1H NMR spectra of thiolatocobalamins. A GSCbl, B NACCbl, C
NACMECbl in D2O, and D D,L-HcyCbl, pD 5.9 (0.1 M MES buffer), 23 ± 1 ºC. Five major signals can be seen, corresponding to the resonances of the B7, B2, B4, R1 and
C10 protons (see Figure 1.2).
44
vacuum. Yield: 145.7 mg (98%). The purity assessed by conversion to
1 dicyanocobalamin(197) was 99 ± 2%. H NMR (D2O, δ, ppm): 5.92 (s C10), 6.15 (d R1),
6.33 (s B4), 6.84 (s B2), 7.07 (s B7) (184). The cobalamin purity assessed by 1H NMR
- - spectroscopy was 98%. ES-MS, m/z: 1408.5 (calcd for [SO3Cbl] , C62H89CoN13O17PS =
1409.5).
2.2.5 Kinetic Measurements
Solutions in the pH range 1.5 - 2.5 were prepared from aqueous solution of
HNO3. Solutions in the pH range 2.5 - 3.9, were prepared using 0.1 M acetate/acetic acid
buffer (pKa = 4.75). The pH values of the solutions were adjusted using a pH meter using
conc. HNO3 and conc. NaOH.
GSCbl or NACMECbl solutions (~0.01 M) were prepared freshly and used within
1 h of preparation. The rates of GSCbl and NACMECbl decomposition were measured
under pseudo first-order conditions in excess [H+]. To a solution of a specific pH, an aliquot of the thiolatocobalamin was added and the absorbance was measured at 350 nm
as a function of time in a Cary 5000 UV/visible spectrophotometer. The ionic strength
was kept constant at 0.5 M using NaNO3.
45
2.3 Results
2.3.1 Synthesis and Characterization of D,L-Homocysteinylcobalamin, N-Acetyl-
L-cysteinylcobalamin (Na+ salt) and 2-N-acetylamino-2-carbomethoxy-L-
ethanethiolatocobalamin
A general procedure for the synthesis of non-alkyl Cbls in aqueous solution is reported. This procedure, which results in high yield and purity of the desired product, consists in the addition of a small excess of the ligand to a highly concentrated solution of
aquacobalamin, followed by the addition of acetone to precipitate the product after
completion of the reaction. This procedure can be utilized to synthesize the novel and
biologically relevant thiolatocobalamin derivatives HcyCbl, Na[NACCbl], and
NACMECbl, in high purity (>95%) and yield (>70%). To obtain a pure product, it was
necessary to synthesize HcyCbl under anaerobic conditions, probably as a consequence
of rapid B12-catalysed aerial oxidation of the thiol reactant (41). All other syntheses were carried out under aerobic conditions. The products were characterized by ES-MS,
1H NMR spectroscopy, and UV/visible spectroscopy. Figure 2.2 gives the UV/visible
spectra of HcyCbl, NACCbl, NACMECbl, and GSCbl. All RSCbl show very similar
electronic spectra, making it impossible to differentiate between them using this
technique. Figure 2.3 gives the 1H NMR spectra of the aromatic region for HcyCbl,
NACCbl, NACMECbl, and GSCbl. The RSCbl spectra show the expected five signals attributable to the B2, B4, B7 protons of the α-5,6-dimethylbenzimidazole nucleotide, the
C10 proton of the corrin ring and the R1 proton of the ribose (Figure 2.3). Since a racemic mixture (D,L-Hcy) of the ligand was used for the HcyCbl synthesis, two
46
stereoisomers of HcyCbl are formed. This is evident from the observation of two peaks at
6.10 ppm (Figure 2.3D). 1H NMR chemical shifts in the aromatic region and UV/visible
wavelength maxima for the new derivatives are summarized in Table 2.1. From this table
it can be seen that there is excellent agreement between the chemical shifts and
wavelength maxima for all RSCbls.
The percentage of cobalamin impurities present in the products can also be
estimated from the aromatic region of the 1H NMR spectrum, and was found to be ~2%
for HcyCbl, Na[NACCbl], and NACMECbl. The percentage of non-corrinoid products
(salts) in the product can be determined by converting the thiolatocobalamin to
-1 -1 dicyanocobalamin (ε367nm = 30.4 mM cm ) (197), and was found to be ≤ 5%.
2.3.2 Acid-catalyzed Decomposition of GSCbl and NACMECbl
Figure 2.4 shows plots of change in the spectra of GSCbl (at pH 1.52) and
NACMECbl (at pH 1.65) as a function of time. The decay of peaks characteristic of
+ thiolatocobalamins (λmax = 333 and 372 nm) and the growth of the H2OCbl peak at 350
nm with sharp isosbestic points (λ = 340 and 363 nm) indicate that the decomposition of
+ these RSCbls in acidic media proceeds cleanly with H2OCbl as the product.
To probe the mechanism for the decomposition of the thiolatocobalamins, the rate
of the decomposition as a function of pH was measured using a Cary 5000 UV/visible
spectrophotometer. A small aliquot (10 - 30 μl) of the RSCbl in water was added to an
acid solution ([H+] ≥ 10 × [RSCbl]). A typical plot of absorbance vs. time for the
decomposition of GSCbl (at pH 1.52) or NACMECbl (at pH 1.65) is shown in Figure 2.5.
The data were fitted to a first-order rate equation, giving a rate constant (kobs) of
47
Table 2.1: UV/visible and 1H NMR spectroscopy data for cobalamins
UV/visible Spectroscopy 1H NMR Spectroscopy Data Data Cobalamin Chemical shift (ppm)
λ max (nm) B7 B2 B4 R1 C10
+ H2OCbl 349 411 525 7.18 6.54 6.47 6.26 6.30
GSCbl 333 372 428 534 7.19 6.95 6.39 6.28 6.09
NACCbl- 333 372 428 534 7.19 6.95 6.40 6.28 6.09
NACMECbl 333 372 428 534 7.19 6.95 6.40 6.28 6.09
HcyCbl 333 372 428 534 7.20 6.95 6.38 6.28 6.10
- SO3Cbl 312 365 418 517 7.17 6.94 6.43 6.25 6.02
NO2Cbl 354 413 532 7.20 6.74 6.42 6.28 6.20
48
A 0.6
0.5
0.4
Abs 0.3
0.2
0.1 320 340 360 380 400 λ (nm) 1.5 B
1.0
Abs 0.5
0.0 320 340 360 380 400 λ (nm)
Figure 2.4. Acid-catalyzed decomposition of thiolatocobalamins. UV/visible spectra were recorded every 1 min. A GSCbl solution in HNO3 (pH 1.52). B NACMECbl
solution in HNO3 (pH 1.65) at 25 °C and 0.5 M ionic strength.
49
A
0.44
0.40 350 nm 0.36 Abs Abs
0.32
0.28 02468101214 B Time (min) 0.95
0.90
0.85
0.80 350nm 0.75 Abs 0.70
0.65
0.60 0246810 Time (min)
Figure 2.5. Kinetic traces for the acid-catalyzed decomposition of RSCbls. The
+ reaction was followed at 350 nm (formation of H2OCbl ) at 25 °C and 0.5 M ionic
strength. A GSCbl solution in HNO3 (pH 1.52). The best fit to a first-order equation gives
-2 -1 kobs = (6.70 ± 0.04) x 10 s . B NACMECbl solution in HNO3 (pH 1.65). The best fit to a
-2 -1 first-order equation gives kobs = (1.58 ± 0.02) x 10 s .
50
-2 -1 -2 -1 (6.70 ± 0.04) x 10 s (t1/2= 104 s) and (1.58 ± 0.02) x 10 s (t1/2= 44 s) for GSCbl and
NACMECbl, respectively.
Figure 2.6 summarizes the dependence of the observed rate constants for the decomposition on pH for GSCbl and NACMECbl. The plots show that the rate of decomposition decreases with increase in pH. This implies that the reaction is acid- dependent. The proposed mechanism is given in Scheme 2.1, where Ka is the acid dissociation constant of the protonated RSCbl and k1 is the rate constant of the decomposition reaction.
Scheme 2.1
+
From Scheme 2.1, it can be shown that
− pH 10 k1 kobs = (2.1) − pH +1010 − pKa
-3 -1 Fitting the data in Figure 2.6 to equation 2.1 gave k1 = (8.3 ± 0.4) x 10 s and
-2 -1 pKa = 2.1 ± 0.1 for GSCbl, and k1 = (3.2 ± 0.2) x 10 s and pKa = 1.5 ± 0.1 for
NACMECbl.
2.3.3 Synthesis and Characterization of Sulfitocobalamin
- SO3Cbl is a biologically important B12 metabolite (145-151), and there are several reports concerning the synthesis and isolation of this derivative (149, 165, 201). These
51
A 0.006 ) -1 0.004 (s obs k 0.002
0.000 1.52.02.53.03.54.04.5 0.025 pH B 0.020
)
-1 0.015 (s obs obs
k 0.010
0.005
0.000 1.0 1.5 2.0 2.5 3.0 3.5 pH
Figure 2.6 Plots of kobs vs pH for the decomposition of RSCbls. A GSCbl and B
-3 -1 NACMECbl. The data were fitted to equation (2.1) giving k1= 8.3 x 10 s and pKa = 2.1
-2 -1 ± 0.1 for GSCbl, and k1 = 3.2 x10 s and pKa = 1.6 ± 0.1 for NACMECbl. All the kobs were calculated at 25 °C and 0.5 M ionic strength.
52
- methods require a large excess of sulfite to obtain complete formation of SO3Cbl , requiring subsequent de-salting of the product using column chromatography. B12-
catalyzed oxidation of sulfite to sulfate can also result in the product being contaminated
by the byproduct sulfatocobalamin (152). It was of interest to see if our procedure could
be applied to the synthesis and isolation of pure Na[SO3Cbl] in high yield, and indeed
this cobalamin was readily synthesized in the presence of air using 1.2 mole equiv. sulfite
without the need for either anaerobic conditions or subsequent desalting by column
chromatography to remove excess sulfite. UV/visible and 1H NMR spectra of
Na[SO3Cbl] are given in Figure 2.7. The latter clearly demonstrates that the product is
pure (~98%). Chemical shift values and UV/visible wavelength maxima for Na[SO3Cbl]
are summarized in Table 2.1. The percentage of salts in the product was found to be ~1%
by conversion to dicyanocobalamin.
2.4 Discussion
In Chapter 2 we reported the synthesis of several novel Cbl derivatives. The syntheses were carried out using our previously reported, very simple procedure for the synthesis of RSCbls. Although equilibria and rate constants for the formation of a wide range of non-alkylcobalamin derivatives in aqueous solution ligated via S, O or N atoms to the cobalt(III) have been reported (181), very few studies report the actual isolation of these compounds in a pure form. The general synthetic method presented in this work has several advantages over previous synthetic procedures. Excess ligand is used to ensure
the reaction goes to completion, requiring a subsequent column chromatography de-
salting step (typically Amberlite XAD-2 or XAD-4) to remove the remaining unreacted
53
A
0.8
0.6
Abs 0.4
0.2
0.0 300 400 500 600 λ (nm) B
7.4 7.2 6.66.87.0 6.4 6.2 6.0 5.8 ppm
Figure 2.7. Characterization of sulfitocobalamin. A UV/visible spectrum of
1 sulfitocobalamin. The spectrum was recorded in H2O at 25 ± 1 ºC. B H NMR spectrum.
The spectrum was recorded in D2O, pD 5.9 (0.1 M MES buffer), 23 ± 1 ºC. Five major
signals are observed at 6.17, 6.94, 6.43, 6.25, and 6.03 ppm, in agreement with literature
values (183).
54
ligand prior to isolation of the product. A Sephadex C-25 column chromatography step may also be required to separate the product from unwanted cobalamin side-products.
The innovation presented in our work is the use of very high, practically saturated, concentrations of aquacobalamin in addition to high ligand concentrations, so that the desired product is rapidly and completely formed using essentially molar equivalents of ligand. The requirement of a lower mole equivalent of ligand for complete formation of
+ the product at higher H2OCbl concentrations is readily understood from the definition of
the formation constant for the complex. Since the product solution is so concentrated, the
product is easily isolated in high yield by the addition of cold acetone. In addition, since high concentrations of ligand are also used, the percentage of ligand that is converted to other forms during the reaction can be significantly reduced, so that excess ligand is not required to compensate for side reactions involving the ligand. For example, the percentage of reduced thiol that undergoes B12-catalyzed aerial oxidation to its inactive
disulfide form (disulfides do not react with aquacobalamin(168)) during the synthesis of
thiolatocobalamins from aquacobalamin is considerably less if the synthesis is carried out
at high concentrations, and can remove the need for a large excess of the reduced thiol
and/or anaerobic conditions.
As reported in Suarez-Moreira et al. (85) the procedure can be used for the
synthesis of RSCbl and other non-alkyl Cbls such as NO2Cbl in high yield and purity. We
anticipate that this procedure can be readily adapted for the synthesis of a wide variety of
non-alkyl cobalamin derivatives, and will thus prove to be extremely useful to those
interested in synthesizing cobalamin derivatives to assist in the identification of unknown
55
cobalamins isolated from biological samples, and those interested in testing the biological activities of different cobalamin derivatives in studies designed to elucidate uptake and intracellular B12 processing mechanisms. Indeed it has already been successfully used by
Hannibal et al. for the synthesis of standards necessary for the identification of intracellular derivatives (50).
The novel compounds HcyCbl, Na[NACCbl], and NACMECbl have been synthesized for the first time in high yield and purity. The Cbls were synthesized using our previously reported, remarkably simple procedure for the synthesis of RSCbls (157).
It has also been demonstrated that this method can be utilized to prepare other non-thiol
containing cobalamin derivatives such as Na[SO3Cbl] in high yield and purity, thus
removing the need for a subsequent column chromatography de-salting step to remove
excess ligand and/or anaerobic conditions to prevent B12-catalyzed oxidation of air-
sensitive ligands. We anticipate that this procedure can be readily adapted for the
synthesis of a wide variety of non-alkyl Cbl derivatives, and will thus prove to be extremely useful to those interested in synthesizing Cbl derivatives to assist in the
identification of unknown cobalamins isolated from biological samples, and those
interested in testing the biological activities of different Cbl derivatives in studies
designed to elucidate uptake and intracellular B12 processing mechanisms.
56
CHAPTER 3
Exploring the Role of Glutathione in Cobalamin Processing in Human Aortic
Endothelial Cells
3.1 Introduction
Although MeCbl and AdoCbl are the main Cbl species isolated from biological tissues (142), other Cbl derivatives have also been isolated, including glutathionylcobalamin (GSCbl) (40, 46-49), nitrocobalamin (NO2Cbl), and
sulfitocobalamin (SO3Cbl) (145, 149). However, the physiological relevance of the observation of these other Cbl derivatives remained, until recently, unclear; that is, are these Cbl derivatives naturally occurring or simply the result of artifactual formation during the extraction and purification procedures? Recent studies in the Jacobsen lab showed that NO2Cbl and SO3Cbl are probably artifacts of the isolation procedure,
whereas GSCbl is a naturally occurring Cbl present in bovine aortic endothelial cells
(BAEC) and in the human hepato carcinoma cell line HepG2 (50).
There is increasing evidence that glutathione (GSH) plays an important role in
intracellular Cbl processing. While our studies were underway, GSH was identified as
being essential for the early reductive dealkylation step in Cbl processing by the
MMAHC protein (30). It has also been suggested that GSCbl is an intermediate in Cbl
cofactor biosynthesis (47) and that GSCbl plays a role in the modulation of inflammation
56 57
(138). Furthermore RSCbls such as GSCbl have potential as therapeutics in the treatment of neuropsychiatric disorders (169).
Whether or not significant amounts of GSCbl are formed in primary human cells or tissues has not yet been reported. Importantly, even though low levels of cystathionine
β-synthase (CBS) activity were observed in human umbilical vein endothelial cells in culture (121), Chen et al. reported that human cardiovascular cells did not express CBS or BHMT (120). Hence cardiovascular cells and tissues are not capable of converting
Hcy to Cys through the transulfuration pathway because they do not express CBS (119).
This finding has two important direct implications to our work. Firstly, cardiovascular cells rely solely on MeCbl-dependent MS to metabolize Hcy (122), and vascular Hcy homeostasis should therefore be particularly sensitive to Cbl levels. Secondly, the inability of cardiovascular cells to convert Met to Cys via the transulfuration pathway provides us with an elegant method to deplete cardiovascular cells of GSH by Cys starvation (Figure 3.1), since GSH synthesis is exclusively dependent on externally added
Cys.
The aim of this work was to identify the naturally occurring Cbl metabolites in primary human aortic endothelial cells (HAEC) and to differentiate these metabolites from Cbl derivatives that are artifactually formed through β-axial ligand exchange during the isolation and purification procedures. We have also sought to clarify the importance of GSH in Cbl metabolism. GSCbl was found to be a naturally occurring Cbl metabolite in HAEC, and evidence supporting a key role of GSH in intracellular Cbl metabolism is provided.
58
Figure 3.1. Key metabolic pathways involved in the synthesis of GSH. Hcy is methylated by B12-dependent methionine synthase (MS) using methyltetrahydrofolate
(MTFH) as the methyl donor. Methionine adenosyl transferase converts Met into S-
adenosylmethionine (SAM). SAM serves as a methyl group donor for reactions
catalyzed by methyl transferases, yielding a methylated product and S-
adenosylhomocysteine (SAH). SAH is hydrolyzed back to Hcy by
adenosylhomocysteine hydrolase. In liver, Hcy can be converted to Cys via the
transulfuration pathway. Hcy is first converted to cystathionine by B6-dependent
cystathionine beta synthase (CBS). In HAEC CBS is inactive. Cystathionine can be
cleaved by cystathionine γ-lyase (CGL) to yield Cys and α-ketobutyrate. Cys is the
limiting substrate for GSH synthesis. Two ATP-dependent synthetases, glutamylcysteine
synthetase (GCS) and glutathione synthetase (GS), catalyze the synthesis of GSH from
Cys, Glu and Gly.
59
60
The Specific Aims of this chapter are:
• To identify naturally occurring Cbl metabolites in HAEC and to
differentiate these from the artifacts generated during the extraction of
Cbls from the cell samples.
• To study the role of GSH in Cbl cofactor biosynthesis using a model of
GSH depletion by Cys starvation.
3.2 Experimental Section
General. Dulbecco’s phosphate buffered saline (DPBS), AdoCbl, MeCbl, CNCbl,
GSH, L-Cys, ascorbic acid, L-Met, Na2SO3, NaNO2 and cystathionine were purchased
from Sigma. HOCbl•HCl was purchased from Fluka. [57Co]-CNCbl (specific activity 379
μCi/mg, 10.5 μCi/mL) was obtained from MP Biomedical (Solon, OH) and (6S)-N5-
methyltetrahydrofolic acid, calcium salt, from Eprova AG, Schaffhausen, Switzerland.
Glacial acetic acid, acetonitrile (both HPLC grade) and ethanol were used without further
purification. GSCbl, NO2Cbl and SO3Cbl were synthesized according to published
methods (85).
For the GSH-depleted HAEC experiments, a stock solution of L-Hcy was freshly
prepared by hydrolyzing L-homocysteine thiolactone (HTL, 153.6 mg) in NaOH (200 μl,
5 M) for 5 min at 37 oC. The resulting solution was neutralized with HCl (200 μl, 5 M), diluted with PBS to 1 ml and kept under argon until use to prevent oxidation. The final total thiol concentration was determined by the Ellman’s method (199).
All errors are reported as one standard deviation of the mean.
61
57 + 57 Synthesis of [ Co]-H2OCbl . [ Co]-CNCbl (2 μCi) was stirred in 20 mM acetic
acid for 2 days under argon bubbling. The product was then passed through a C18 Sep-
Pak column (Waters Assoc. Milford, MA), which was activated with CH3CN (1 ml) and
washed with H2O (6 ml). The sample was loaded, washed with H2O (3 ml) and the
product eluted with CH3CN (1 ml). The eluate was taken to dryness, re-suspended in H2O
(100 μl) and purified by HPLC.
HPLC chromatography. HPLC were carried out using an Agilent 1100 system
equipped with a Zorbax SB C-18 column (4.6 x 250 mm, 5 µm particle size, Agilent), and a Zorbax SB-C18 analytical guard column (4.6 x 12.5 mm, 5 μm). The mobile phase
consisted of a mixture of 0.1% acetic acid/acetate buffer titrated to pH 3.50 with NH4OH
(Solvent A), and acetonitrile containing 0.1% acetic acid (Solvent B). The conditions were as follows: 5% B from 0-2 min; 5 to 1% B from 2-14 min; 15 to 18% B from 14-19 min; 18 to 35% B from 19-32 min; 60% B 32-33 min; 5% B 33-35 min. A flow rate of 1 mL/min was used and all gradients were linear.
Expression of human recombinant transcobalamin. Human recombinant transcobalamin (hrTC) was produced in a baculovirus expression system using
Trichoplusia ni cells (202). The culture supernatant was enriched by ion exchange column chromatography using CM-Sephadex C-50. The cell culture supernatant (25 ml) was applied to a 5 ml CM-Sephadex column pre-equilibrated with 0.02 M Na2HPO4/ 0.1
M NaCl at pH 5.4 and the protein eluted with 0.1 M Na2HPO4 containing 1 M NaCl at
pH 5.8. Fractions (5 ml) were collected and the protein elution was followed
62
spectrophotometrically at 280 nm. An aliquot of each fraction was assayed for apo-TC by the unsaturated cobalamin-binding capacity assay (203).
Endothelial cell culture. HAEC were harvested by collagenase treatment of sterile segments from non-atherosclerotic thoracic aorta (discarded tissue) obtained from heart transplant donors. Each endothelial cell isolate was stored and passaged separately.
HAEC were maintained in complete M199 (Cbl free) medium plus 2% FBS in a humidified 95% air, 5% CO2 incubator at 37 ˚C. The medium was supplemented with
endothelial cell growth supplements (Cambrex). The cells were passaged with trypsin at a
ratio of 1:3 and grown on fibronectin-coated flasks. For most experiments the cells were
used up to passage 6. The DNA content of cell extracts was determined using the
CyQuant Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s
specifications.
Extraction and identification of Cbl derivatives in HAEC. For assessing
intracellular Cbl metabolites, HAEC were cultured in T-175 culture flasks until ~80%
confluent (5 x 106 cells/flask). [57Co]-CNCbl complexed with TC ([57Co]-CNCbl-TC,
1:1000 [57Co]-CNCbl:TC) was added to the medium at a final concentration of 195 pM
(0.1 μCi/ml) and the culture incubated for 24 h. Transcobalamin was complexed with Cbl
to facilitate uptake by HAEC by receptor-mediated endocytosis. Cbls were extracted
according to published methods with some modifications (47). Specifically, cells were
harvested and re-suspended in extraction buffer (Tris/HCl 50 mM pH 7.4, NaCl 150 mM,
2% Triton X100). Ethanol (1 ml) was added to the cell extract and the extract left
standing at room temperature for 20 min. The samples were centrifuged at 10,000 rpm for
63
10 min, and the supernatant removed and taken to dryness. The product was washed twice with acetone (500 μl). The precipitate was re-suspended in water and the resulting
Cbl extract spiked with non-radioactive Cbl standards and analyzed by HPLC. Fractions were collected and the radioactivity measured in a gamma counter (Gamma 4000,
BECKMAN-Coulter). The radioactivity chromatogram was overlapped with the
UV/visible chromatogram and the presence of radioactivity at the elution time of a cold
Cbl standard taken as proof of that specific Cbl being extracted from HAEC.
The baseline radioactivity level was determined by counting the radioactivity of
60 fractions from a blank run (100 μl H2O) and determining its 99% confidence interval.
In each run, the baseline radioactivity was established by comparing the peak-free
regions of the chromatogram with the baseline radioactivity of the blank run. Each
fraction was compared with the baseline using a one-tailed t-test. The presence of
radioactivity in a fraction was considered positive when the value was statistically greater
than the baseline at p<0.01.
In another set of experiments, Cbl derivatives were extracted as above from
+ + HAEC lysates in the presence of 8 mM non-radioactive H2OCbl . Excess H2OCbl traps free ligands such as GSH, nitrite and sulfite as their corresponding Cbl forms (GSCbl,
NO2Cbl and SO3Cbl, respectively), and hence prevents these ligands from reacting with
57 + [ Co]-H2OCbl released from the cells upon cell lysis.
+ Determining whether H2OCbl reacts with GSH during the extraction procedure
+ to form GSCbl. To assess whether GSCbl is potentially formed from H2OCbl reacting with GSH during the extraction and purification procedure, HAEC were harvested and
64
57 + re-suspended in lysis buffer containing 10 nCi of [ Co]-H2OCbl . Cbls were extracted as
described above and the presence of GSCbl in the final extract determined as described in
the previous section.
Determining the percentage of corrinoids other than Cbls by conversion to
CNCbl. After the total Cbl extraction was carried out, samples were taken to dryness and
resuspended in 10 molar equiv. KCN. The mixture was allowed to react at room
temperature for 1 h under illumination, resulting in all Cbls being converted to
dicyanocobalamin, (CN)2Cbl. Acidifying the sample with 1% acetic acid and taking it to
dryness removes the α-cyano group, giving CNCbl. Finally the sample was re-suspended
in water and subjected to HPLC analysis.
Depleting HAEC of GSH. A cysteine-free modified MCDB 105 medium was
prepared that did not contain L-Cys, L-methionine, vitamin B12, or folate. L-methionine
(3.2 mM) and tetrahydrofolate (50 nM in 10 mM ascorbate) were added to the medium
and the medium supplemented with glycine (15 mg/l), heparin (90 mg/l), potassium
chloride (15 mg/l), endothelial cell growth factors (Cambrex, 150 mg/l) and 15% FBS.
HAEC were grown in complete MCDB 105 medium until ~80% confluent. The medium was replaced with L-Cys-free MCDB 105 medium. Cells were harvested at several time points and intracellular L-Cys and GSH concentrations determined (see below). For the determination of intracellular Cbls of GSH-depleted HAEC, the modified MCDB 105 medium was supplemented with glycine (15 mg/l), heparin (90 mg/l), potassium chloride
(15 mg/l), endothelial cell growth factors and 2% FBS.
65
Thiol concentrations were determined with an HPLC-based method.(204-206).
Briefly, 5 μl of amyl alcohol was added to cell lysates (100 μl) that were treated with sodium borohydride in 0.1 N NaOH to reduce thiols to their sulfhydryl forms. The solution was neutralized with HCl and the proteins precipitated with perchloric acid (20
μL, 5 M). The sample was cooled (ice bath, 10 min), centrifuged and the thiol-containing supernatant derivatized with monobromobimane (10 μl, 50 mM). The different thiol- bimane derivatives were resolved by HPLC on a C-18 column with fluorescence detection. The thiol concentration was determined by taking the area for the thiol-bimane peak and calculating its concentration using a regression equation derived from a standard curve.
Extraction and identification of Cbl derivatives in GSH-depleted HAEC. HAEC were grown in M199 medium until ~80% confluent and labeled with [57Co]-CNCbl-TC
(0.1 μCi) for 24 h as described above. The cells were washed with PBS (x 3) and further incubated for 20 h with L-Cys free modified MCDB 105 medium. The medium was prepared as described above but supplemented with Cambrex EGM-2 supplements and
2% FBS to mimic the growth conditions the cells had been exposed to. The cells were harvested and the Cbls extracted and analyzed by HPLC.
3.3 Results
3.3.1 Extraction and identification of intracellular Cbl derivatives in HAEC
Intracellular Cbl concentrations are extremely low (pM), hence a convenient method to determine intracellular Cbl forms is to incubate cells with radioactive [57Co]-
66
CNCbl, allowing sufficient time for uptake and intracellular conversion of [57Co]-CNCbl
to other Cbl forms, extract the intracellular Cbls, and quantify them by co-injecting with
Cbl standards on a HPLC instrument. Fractions are collected, the radioactivity measured,
and the radioactivity chromatogram overlapped with the UV/visible chromatogram of the
Cbl standards. The presence of radioactivity at the elution time of a cold Cbl standard is taken as proof of that specific Cbl being extracted (50).
Of particular interest is the role of GSH in intracellular Cbl processing. Thus, an important question was to examine whether significant amounts of GSCbl exist intracellularly in HAEC. The first step was to optimize the isolation procedure to minimize GSCbl loss. The standard procedure for extracting and purifying Cbls from biological samples includes an ethanol extraction step at 80 ºC, taking the supernatant to dryness and washing the product with acetone to remove lipids. When the extraction procedure was carried out on HAEC extracts spiked with GSCbl (25 nmol), identification of the Cbl products by HPLC showed that only 2.5 ± 1.8 % (N = 3) of the radioactivity co-eluted with authentic GSCbl (Figure 3.2). Based on the low recovery of authentic
+ GSCbl, we suspected that GSCbl had decomposed to H2OCbl during the hot ethanol extraction step, and indeed, the remaining recovered radioactivity was associated with
+ H2OCbl , in agreement with results by Hannibal et al (50). Therefore, the temperature of extraction was reduced from 80 ºC to room temperature (RT). Consistent with this hypothesis, carrying out the entire procedure with a room temperature ethanol extraction step led to a 10-fold increase (18.6 ± 0.2 %, N = 3) in the amount of GSCbl recovered
67
20 18 16 14 12 10 8 6 4 GSCbl recovered (%) 2 0 RT 80 °C
Figure 3.2. GSCbl recovery. Confluent HAEC were harvested and 25 nmol of pure
GSCbl was added to the lysis buffer. The extraction procedure was carried out at room temperature (RT) or at 80 °C. The final extract was analyzed by HPLC. Bars represent mean ± SD, N=3.
68
(Figure 3.2). The ethanol extraction step was therefore carried out at room temperature for all subsequent experiments.
To determine whether GSCbl (and other non-alkylCbl derivatives) exists intracellularly in HAEC, the optimal time required to incubate HAEC with [57Co]-
CNCbl-TC to obtain maximum cell-associated radioactivity was established. HAEC were
exposed to [57Co]-CNCbl-TC (164 pM, 0.084 μCi/ml) for a range of times. At different time points cells were harvested, washed with PBS, and the cell-associated radioactivity
determined using a gamma counter. As shown in Figure 3.3, a steady time-dependent
increase in cell-associated [57Co]-CNCbl-TC over 24 h was observed. Incubation for 48 h resulted in a ~40% reduction of cell-associated radioactivity; therefore, cells were incubated with [57Co]-CNCbl-TC for 24 h for all subsequent experiments.
To determine important intracellular Cbl derivatives, HAEC were incubated for
24 h in [57Co]-CNCbl-TC-supplemented medium, harvested, and the Cbls extracted and
purified. For identification purposes, the Cbl extract was spiked with non-radioactive Cbl
+ standards (H2OCbl , CNCbl, NO2Cbl, GSCbl, MeCbl and AdoCbl) and the extract separated by reverse-phase HPLC. The amount of each Cbl was determined by measuring
the radioactivity in each fraction. A representative HPLC chromatogram of the Cbl
extract is given in Figure 3.4, which shows an overlay of the UV/visible spectrum of the
non-radioactive Cbl standards (light gray trace) with the radioactivity of the fractions
(black trace). These data indicate that HAEC are able to synthesize AdoCbl and MeCbl
from the CNCbl precursor under the growth conditions. Smaller amounts of [57Co]-
+ 57 H2OCbl , GSCbl and NO2Cbl were also isolated, as was unprocessed [ Co]-CNCbl. The
69
16 14 12 10 8 6 4 fmol Cbl / µg DNA fmol Cbl / µg 2 0 4 8 13 17 24 Hours
Figure 3.3. Cbl uptake by HAEC. Measure of intracellular Cbl concentration (fmol
Cbl / μg DNA) after incubation with [57Co]-CNCbl-TC at various time points. At the
indicated times the cells were washed, harvested and the amount of Cbl incorporated in
the cells determined by measuring the cell-associated radioactivity. Bars represent mean
± SD, N=3.
70
3000 80
70 2500
60 ) ( cpm AdoCbl 2000
( ) 50 1500
254 nm 254
40 + MeCbl - Cbl
30 GSCbl 2 OCbl 1000 2 mAU Cbl 3 H 20 NO CNCbl CNCbl SO 500 10
0 0 12 14 16 18 20 22 24 26 28 Time (min)
Figure 3.4. Cobalamin profile in HAEC. Cells were labeled with [57Co]CNCbl-TC for
24 h, and the Cbls extracted at RT. The extract was spiked with non-radioactive Cbl
standards and subjected to HPLC analysis. Fractions were collected and counted in a γ counter. The overlap of the radioactive chromatogram ( ) with the pure standards ( ) indicates the presence of that Cbl in the cell extract.
71
Table 3.1. Percentages of cobalamins extracted from HAEC in the absence or presence of an added free ligand trap in the cell lysis buffer, and after GSH depletion by L-Cys starvation. ND = not detectable.
% Cbl extracted in % Cbl extracted in % Cbl extracted after the absence of a the presence of a GSH depletion in the ligand trap ligand trap absence of a ligand (mean ± SD, N=7) (mean ± SD, N=3) trap (mean ± SD, N=3)
+ H2OCbl 12.6 ± 6.0 11.7 ± 1.6 7.2 ± 0.2
GSCbl 5.8 ± 3.6 1.2 ± 0.7 ND
CNCbl 24 ± 11 31 ± 19 ND
AdoCbl 19.3 ± 7.5 40 ± 13 72.1 ± 3.3
MeCbl 7.9 ± 3.7 15.4 ± 5.2 12.7 ± 0.4
SO3Cbl* 0.7 ± 0.2 ND ND
NO2Cbl* 6.1 ± 1.1 ND 4.5 ± 1.8
Other 24 ± 18 0.5 ± 0.9 3.3 ± 1.7
* For these derivatives, N=3 in column 1.
72
first column of Table 3.1 summarizes the results (N = 7). The major Cbls extracted were
+ AdoCbl (19.3 ± 7.5%), MeCbl (7.9 ± 3.7%), H2OCbl (12.6 ± 6.0%) and unprocessed
CNCbl (24 ± 11%). Importantly, significant amounts of GSCbl (5.8 ± 3.6%) were also
extracted from the HAEC, as was NO2Cbl (6.1 ± 1.1%) and SO3Cbl (0.7 ± 0.2%). Table
3.1 also shows that a substantial portion of the radioactivity was associated with
unidentified species (30 ± 18%). To determine whether these identified species were
cobalamins or other corrinoids, HAEC were incubated with [57Co]-CNCbl-TC, the Cbls
extracted, and the extract treated with excess KCN to convert all Cbls to CNCbl. This
sample was then analyzed by HPLC. Most (~90%) of the radioactivity co-eluted with the
CNCbl peak. However ~10% of radioactivity was associated with unidentified fractions.
No further attempts were made to identify these species.
3.3.2 Extraction and Identification of Intracellular Cbl derivatives in HAEC in the
presence of a Free Ligand Trap
57 + From Table 3.1 it can be seen that significant amounts of [ Co]-H2OCbl are
extracted from HAEC incubated with 57Co-CNCbl (column 1). This is consistent with
similar findings in other mammalian cell types (196, 207). Cbl(I) and Cbl(II) are rapidly
+ converted to H2OCbl in the presence of air (208, 209), thus these intracellular forms will
+ be oxidized to form H2OCbl during the aerobic extraction process (Figure 3.5). The β-
+ + axial ligand of H2OCbl is rapidly substituted by stronger nucleophiles. H2OCbl can
therefore potentially react with free ligands released from the cell (including GSH) to
form the corresponding Cbl derivatives during the extraction and purification procedures
73
GS Co(III) N GSH
Co(I) NO2 - N O2 NO2 Co(III)
OH2 N
Co(III) - SO3 SO3 O N 2 Co(III) Co(II) Nucleophile N N N OH2 Co(III) Co(III) N N (intracellular)
Figure 3.5. Representation of β-axial ligand exchange reactions. Intracellular Cbl(I)
+ and Cbl(II) are oxidized in the presence of O2 after cell lysis to yield H2OCbl . This
+ + + H2OCbl , in addition to intracellular H2OCbl results in a pool of H2OCbl in the lysate
that can react with different nuleophiles released from the lysed cells to yield artifactual
Cbl derivatives.
74
(Figure 3.5). Thus, the datain Table 3.1, column 1, may not truly reflect the intracellular
Cbl profile but could instead be a consequence of β-axial exchange reactions occurring
during the extraction and purification procedures (Figure 3.6).
As an initial step to distinguish between these possibilities, HAEC were lysed in
57 + the presence of [ Co]-H2OCbl (10 nCi) without prior exposure to Cbl, and the
extraction and HPLC separation protocols performed. Figure 3.7 shows a typical
57 + chromatogram comparing the Cbl profile obtained when [ Co]-H2OCbl (10 nCi) was
added at the time of lysis (which did not allow for uptake and processing) to that obtained
57 + with the addition of [ Co]-H2OCbl (10 nCi) 24 h prior to the lysis step (to facilitate
57 + uptake and processing). Importantly, when radioactive [ Co]-H2OCbl was added at the time of lysis, a substantial amount (12.2 ± 0.5%, N=3) of the total radioactivity was
recovered as 57Co-GSCbl, whereas only ~30% of the radioactivity was recovered as 57Co-
+ H2OCbl . The remaining reactivity was presumably associated with other derivatives such as NO2Cbl, SO3Cbl, as was previously observed for BAEC (50). Hence, it was
+ concluded that intracellular Cbl(I)/Cbl(II)/H2OCbl can indeed react with free GSH, which can only have been derived intracellularly, to form GSCbl during the extraction/purification procedures (Figure 3.6).
Since it was established that the observation of GSCbl could be simply due to an
+ extracellular reaction of H2OCbl with GSH to form GSCbl within the isolated milieu, it
was sought to competitively inhibit this reaction by adding an excess of a free ligand trap
to obtain a more accurate reflection of the intracellular composition of Cbl derivatives.
+ Excess non-radioactive H2OCbl was added to the lysis buffer to trap GSH and other free
75
Protein-bound Cbl(II)
X artifactual pathway
processing
Artifactual + intracellular GSCbl
Figure 3.6. Intracellular and artifactual pathways for GSCbl formation. GSCbl could be formed inside the cell by the processing of various Cbl derivatives. On the other
+ hand, H2OCbl present in the cell or a product of intracellular Cbl(I) and Cbl(II)
oxidation could also react with free GSH released upon cell lysis, yielding artifactual
GSCbl.
76
1000 1000
no processing after 24 hour cell processing
800 800 cpm ( , ) AdoCbl AdoCbl
600 600 ( )
+ MeCbl MeCbl
254nm 400 400 OCbl 2 H GSCbl GSCbl mAU 200 200
0 0 10 20 30 40 Time (mn) Figure 3.7. Artifactual formation of Cbl derivatives in HAEC. Cells were labeled
57 + with [ Co]H2OCbl -TC for 24 h or spiked just before lysis, prior to Cbl extraction. The
extract was spiked with non-radioactive Cbl standards and subjected to HPLC analysis.
Fractions were collected from 8 – 13 min and from 30 – 38 min and counted in a γ
counter. The overlap of the radioactive chromatogram ( , ) with a pure Cbl standard peak ( ) indicates the presence of that Cbl in the cell extract. 77
ligands released from HAEC upon lysis (Figure 3.8), prior to isolation and analysis of the
Cbl extract. HAEC were incubated for 24 h in the presence of [57Co]-CNCbl-TC, and the
+ cells harvested and lysed in the presence of non-radioactive H2OCbl . Non-radioactive
+ H2OCbl was added to the lysates in excess (8 mM) compared with the GSH
concentration after cell lysis which was determined to be 0.6 mM ± 0.3 mM. The
concentration of other potential nucleophiles including nitrite and sulfite was
considerably less than this. A typical HPLC chromatogram showing the UV/visible
spectrum overlaid with the radioactivity of the fractions for the Cbl extract is given in
57 57 57 + Figure 3.9, which shows that in addition to Co-MeCbl, Co-AdoCbl, Co-H2OCbl and unprocessed 57Co-CNCbl, 57Co-GSCbl is clearly present in the extract, albeit in much
smaller amounts as compared to extraction in the absence of the ligand trap.
The Cbl profile obtained in the presence of the ligand trap are summarized in
Table 3.1 (column 2). We found that in the presence of the free ligand trap, 1.2 ± 0.7%
GSCbl was extracted. There were no statistical differences between the percentages of
MeCbl and AdoCbl isolated in the absence and presence of the ligand trap as expected,
given that alkylcobalamins do not readily undergo β-axial ligand exchange reactions (50).
Note that unlike the experiments carried out in the absence of a ligand trap, significant
57 57 amounts of Co-NO2Cbl and Co-SO3Cbl were not isolated. Therefore, it is likely that
the presence of both of these species in the extracted product in the absence of the ligand
trap was a consequence of rapid reactions between released intracellular 57Co-
+ H2OCbl /Cbl(II)/Cbl(I) with nitrite or sulfite, respectively, during the extraction/isolation procedure. This agrees with model studies that show formation of NO2Cbl or SO3Cbl
78
intracellular GSCbl (radioactive)
free ligand trap artifactual GSCbl (non-radioactive)
Figure 3.8. Representation of the free ligand trap methodology. After the cells were incubated with [57Co]-CNCbl, the intracellular pool of cobalamin was radioactively
+ labelled. The free ligand trap, an excess of non-radioactive H2OCbl added during the cell lysis, rapidly reacts with released free nucleophiles, such as GSH. This prevents the
+ free nucleophiles from reacting with radioactively labelled intracellular H2OCbl released during the cell lysis.
79
Figure 3.9. Cobalamin profile of HAEC in the presence of a ligand trap. A. Cells were labeled with [57Co]CNCbl-TC for 24 h, and the Cbls extracted in the presence of a
+ free ligand trap (excess non-radioactive H2OCbl , 8 mM) in the lysis buffer. The extract was spiked with non-radioactive Cbl standards and subjected to HPLC analysis. Fractions were collected and counted in a γ counter. The overlap of the radioactive chromatogram
( ) with the pure standards ( ) indicates the presence of that Cbl in the cell extract. B.
Expanded view from 11 – 16 min.
80
A
140 1600
120 1400
CNCbl CNCbl 1200
100 cpm ) (
( ) 1000 80
GSCbl
AdoCbl 800 + 254nm 60 H2OCbl 600 MeCbl
mAU 40 400
20 200
0 0 12 14 16 18 20 22 24 26 28
Time (min)
B 1200 400
1000 350
800 + 300 H2OCbl cpm ( ) ( ) ( GSCbl 600 250
254nm 400 200 mAU
200 150
0 100 11 12 13 14 15 16
Time (min)
81
+ resulting from the reaction between H2OCbl and nitrite or sulfite is rapid and thermodynamically favorable (86, 185). Furthermore, the total radioactivity associated
with unknown species was reduced to 0.5% ± 0.9% as compared to 30 ± 18% in the
absence of the ligand trap (Table 3.1). The lack of detectable amounts of NO2Cbl and
SO3Cbl combined with the dramatic reduction in the percentage of unidentified Cbl
+ species supports the need for addition of excess H2OCbl to efficiently trap free ligands including GSH to obtain accurate levels of intracellular Cbls. This leads to a reduction in the isolation of artifactual Cbl derivatives and confirms that that GSCbl is indeed formed and present intracellularly in HAEC. Moreover, the low recovery of authentic GSCbl even after modifying the extraction conditions observed in this work (19%) and by
Hannibal et al. for BAEC (35%) (50) suggests that the percentage of GSCbl isolated is an underestimation of actual intracellular GSCbl levels.
3.3.3 Determination of Intracellular Cobalamins in GSH-depleted HAEC
HAEC are an ideal cell type to investigate whether or not intracellular GSH levels affect intracellular Cbl processing, since it was previously shown that HAEC do not express CBS (120) and are therefore not able to convert L-methionine to L-Cys. Hence, upon L-Cys starvation, the intracellular GSH levels in HAEC would be expected to decline dramatically, given that L-Cys is the direct intracellular precursor of GSH (Figure
3.1). Previous unpublished results from the Jacobsen lab carried out by Eumelia Tipa confirm that HAEC do not have a functional transulfuration pathway. Therefore, L-Cys starvation should result in a depletion of the GSH pool (Figure 3.1). Tipa grew HAEC in media lacking L-Cys and determined GSH levels at 4, 8, 12, 15, and 18 h. Although
82
intracellular L-Cys levels were relatively unchanged, there was a dramatic decrease in intracellular levels of GSH, with practically negligible intracellular amounts of GSH (30 nmol/mg to 0.5 nmol/mg protein) obtained after 15 h (Figure 3.10).
Based on these results showing that incubation in L-Cys-free medium for 20 h was sufficient to deplete GSH in HAEC, HAEC were initially grown in L-Cys-free medium for 24 h followed by incubation with [57Co]-CNCbl (0.1 mCi) for 24 h. This growth protocol unexpectedly resulted in a 80-90% reduction in cell-associated radioactivity
compared to that of HAEC cultured in L-Cys containing medium, suggesting that Cbl
uptake may be reduced and/or exported into the medium may be increased under these
growth conditions. Because this would preclude the accurate determination of
intracellular Cbls, the growth and Cbl labeling protocol was changed. HAEC were
instead cultured in regular, L-Cys-containing M199 medium until ~80% confluent
followed by addition of 0.1 μCi [57Co]CNCbl-TC. After 24 h, the medium was changed
to fresh L-Cys-free MCDB and the cells incubated for a further 20 h. After this time
period the cells were found to have ~30-50% less cell-associated radioactivity compared
with HAEC grown in regular medium in the presence of [57Co]-CNCbl-TC; however this
level of radioactivity was sufficient for extraction and identification of the intracellular
Cbls. In these experiments HAEC were lysed in the absence of excess non-radioactive
+ H2OCbl in the lysis buffer. A typical chromatogram is shown in Figure 3.11. Table 3.1 gives the percentage of the Cbl forms isolated from HAEC after transfer to a L-Cys- deficient cell medium subsequent to 57Co-CNCbl uptake. The GSH content was assessed prior to the Cbl profiling. Given that the cells were deficient in GSH, it was not
83
35
30
25
20
15
10
5 Thiols (nmol/mg protein) 0 0 2 4 6 8 10 12 14 16 18 20 Time (h)
Figure 3.10. Intracellular L-Cys and GSH concentration in HAEC in response to L-
Cys starvation as a function of time. HAEC were grown until ~80% confluent in complete MCDB 105 medium prior to being transferred to L-Cys-free MCDB medium.
Cells were harvested at various time points and analyzed for intracellular Cys (c) and
GSH (○) content. (Reprinted with permission of Dr. Donald Jacobsen.)
84
3000 80
2500 70 mAU 60 Cbl CNCbl CNCbl + 2000 2 254 nm -
NO 50 GSCbl OCbl 2
1500 Cbl 3
H 40 ( ) ( SO AdoCbl cpm ( ) 1000 30
MeCbl 20 500 10
0 0 10 15 20 25 30 Time (min)
Figure 3.11. Cobalamin profile in GSH-depleted HAEC. Cells were labeled with
[57Co]CNCbl-TC for 24 h, the medium changed to Cys-free medium for 20 h and the
cobalamin profile determined. The extract was spiked with non-radioactive Cbl standards
and subjected to HPLC analysis. Fractions were collected and counted in a γ counter. The
overlap of the radioactive chromatogram ( ) with a pure Cbl standard peak ( ) indicates
the presence of that Cbl in the cell extract.
85
unexpected that [57Co]-GSCbl was undetectable. Remarkably, unprocessed 57Co-CNCbl
was not recovered from the cells, which suggests that upon becoming deficient in GSH,
HAEC expel CNCbl into the medium. The presence of CNCbl in the medium was
confirmed by HPLC. Furthermore, the vast majority of Cbl species detected in the extract
was AdoCbl, with an increased AdoCbl/MeCbl ratio of 5.7 ± 0.3 (N=3) as compared with
a ratio of 2.6 ± 0.3 (N=10) in HAEC which were not GSH-depleted.
3.4 Discussion
The intracellular Cbl profile of HAEC has been determined under normal and
GSH-depleted conditions. The identification of Cbl derivatives in biological samples is of fundamental relevance to the mechanism of Cbl metabolism, cofactor biosynthesis and potential non-cofactor roles of Cbls. However, the identification of naturally occurring
Cbl derivatives poses several challenges. Of primary concern is the limit of detection of the analytical methods used for Cbl analysis, given the low Cbl concentrations found in biological samples. The simplest way to overcome this problem is to use radioactive
[57Co]-Cbl in order to readily detect Cbl levels. Other more involved approaches include
mass spectrometry methods or microbiological detection (210, 211). These methods do
not require radioactive labeling of the material; thus they are better suited to analyze the
Cbl profile from human samples. However, in these studies, use of a cell model permited
the use of [57Co]-Cbl.
As discussed previously in this dissertation, a range of Cbl derivatives, including
SO3Cbl (143-145), NO2Cbl (145, 152), and GSCbl (47-49) have been isolated from
+ mammalian cells and foods. However, non-alkyl cobalamins such as H2OCbl readily
86
undergo β-ligand exchange in which the β-axial ligand is substituted by a stronger
- - - nucleophile such as NO2 , SO3 or GS , forming NO2Cbl, SO3Cbl and GSCbl,
respectively. Indeed, β-ligand exchange reactions have been long recognized and even
used as a means to detect and quantify nucleophiles in biological materials. For instance
+ - the reaction between H2OCbl and SO3 to form SO3Cbl has been used to detect sulfites
in foods (146). However, β-ligand exchange has been often ignored when analyzing
naturally occurring Cbl derivatives. These substitution reactions are independent of
cellular processing; hence metabolites formed through this mechanism have no relevance
to Cbl metabolism. In order to determine whether β-axial exchange affect Cbl profile, a
+ ligand trap (non-radioactive H2OCbl ) was added to the cell lysis buffer. The use of this methodology to differentiate naturally occurring metabolites from artifacts resulting from
β-ligand exchange will be an excellent tool for Cbl researchers.
The presence of GSCbl in the Cbl profile when the free ligand trap was used
(Table 3.1) unequivocally demonstrates for the first time that GSCbl is a naturally
occurring Cbl metabolite present in primary human cells. The fact that most researchers
have not isolated GSCbl from human biological material is likely to be due to the use of aggressive extraction procedures that promote GSCbl decomposition. It was shown that
+ under hot ethanolic extraction conditions, GSCbl decomposes to yield H2OCbl (recovery
< 2 %). Even with our mild extraction procedure, 80% of the GSCbl decomposed to
+ H2OCbl , which suggests that the amount determined in HAEC is underestimated.
It has been proposed that GSCbl (or a closely related thiolatocobalamin adduct) is a precursor of the two coenzyme forms of vitamin B12, AdoCbl and MeCbl (47). MS has
87
also been shown to possess intrinsic thiol oxidase activity (22). Given that thiols can reduce Cbl(III) to yield Cbl(I) (41), this suggests that GSCbl is potentially involved in
Cbl cofactor biosynthesis. While these studies were in progress, the mystery of how the cyano β-axial ligand is removed was solved by Kim et al (33). They showed that
MMACHC catalyzes the reductive decyanation of CNCbl to yield Cbl(II). GSH was not necessary for MMACHC decyanase activity in vitro nor was it capable of acting as an electron donor. However, the enzyme was subsequently shown to catalyze the GSH- dependent reductive de-alkylation of alkylCbl in vitro (30). Moreover, Hannibal et al (32) showed that fibroblasts belonging to the cblC complementation group failed to process alkylCbls for cofactor biosynthesis. Patients belonging to the cblC complementation group bear mutations in the MMACHC gene, which suggests that this protein is indeed involved in the in vivo β-axial ligand removal, an early step in cofactor biosynthesis, and that GSH is essential for this step. Although GSCbl is not an intermediate, the involvement of GSCbl in intracellular Cbl processing cannot be totally discarded at this point.
The effect of GSH depletion on Cbl processing was also assessed. Cbl metabolism was affected in multiple ways. Firstly, the total intracellular Cbl pool was significantly reduced in GSH-depleted HAEC. It was not possible to directly label GSH- depleted cells with [57Co]-CNCbl due to either impaired uptake, impaired retention
capacity or impaired metabolism. HAEC were instead cultured in the presence of
[57Co]CNCbl prior to depleting the cells of GSH by Cys starvation. Even this latter
experimental approach resulted in a decreased intracellular Cbl pool (30-50%) compared
88
with normal GSH conditions. Secondly, GSCbl was not detected in Cbl extracts from
GSH-depleted cells, as expected. Thirdly, an unexpected shift in the AdoCbl/MeCbl ratio was observed. The percentage of AdoCbl in the GSH-depleted cells was actually increased 4-fold (40% in non-depleted cells vs 72% in GSH-depleted cells). This change could either be due to a relative decrease in MeCbl or a relative increase in AdoCbl.
However, since the total amount of intracellular Cbls was significantly decreased (30-
50%), the actual total amount of AdoCbl fell 1.5 fold. Although the percentage of MeCbl remained constant (15% in non-depleted cells vs 13% in GSH-depleted cells), the total amount of MeCbl fell 3-fold. Therefore, depleting HAEC of GSH appears to affect the
MeCbl pool more than the AdoCbl pool.
Low levels of GSH promote a pro-oxidant environment in the cell. During the catalytic cycle of MS, Cbl(I) is formed which is readily oxidized to Cbl(II). This could explain a more significant loss of MeCbl due to a lower stability of the MeCbl cofactor compared to AdoCbl. Another possibility is that the biosynthesis of MeCbl is affected to a greater extent by the oxidative environment compared to AdoCbl biosynthesis. Also of relevance are two studies on effects of MeCbl and oxidative stress in autistic children.
Decreased SAM/SAH and GSH/GSSG ratios were observed that were reverted with folate, betaine and MeCbl supplementation (212, 213). The low GSH/GSSG ratio indicates a state of oxidative stress. The impaired SAM/SAH reveals an impaired methylation potential, probably due to an impaired MeCbl-dependent MS activity since these children also showed mild hyperhomocysteinemia. These data are reminiscent of our HAEC system in that oxidative stress affects MeCbl metabolism.
89
Others have also reported that oxidative stress affects Cbl metabolism, and the association between oxidative stress and Cbl deficiency (163, 214-216). Furthermore Cbl deficiency, either nutritional (217) or due to metabolic impairment (218), is associated with elevated markers of oxidative stress. Impaired Cbl metabolism under oxidative stress conditions and the GSH requirement for general Cbl processing (30) involving the
MMACHC protein also suggest that the synthesis of AdoCbl should be reduced under oxidative stress conditions.
To summarize we have accurately determined the intracellular Cbl profile in primary human vascular cells and have identified GSCbl as a naturally occurring metabolite. Comparing the normal Cbl profile with that obtained for GSH-depleted cells shows that profound changes in Cbl metabolism, most noticeably a substantial change in the AdoCbl/MeCbl ratio, occurs in GSH-depleted HAEC.
90
CHAPTER 4
Vitamin B12 Protects against Homocysteine-Induced Cell Injury in Human Aortic
Endothelial Cells
4.1 Introduction
Hcy is an independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease (219). High plasma levels of Hcy cause adverse effects on the vascular endothelium and can lead to endothelial dysfunction (220). In addition there is evidence that Hcy plays a causal role in atherogenesis and progression of atherosclerosis
(219). Hcy activates the endothelium, leading to increased monocyte cell adhesion (221) and induction of pro-inflammatory cytokines including MCP-1 and IL-8 (222).
Individuals who suffer from hereditary hyperhomocysteinemia have premature thromboembolic complications that are often fatal before the age of 30 (223, 224). Hcy induces endothelial cell death in human cell systems (225-227). The mechanisms by which Hcy exerts its detrimental effects on the endothelium are not completely understood; however, it has been shown that Hcy induces an increase in reactive oxygen species (ROS) levels (reviewed by Papatheodorou et al.)(228) in
90 91
different cell types including human aortic endothelial cells (HAEC) (229).
The mechanisms by which Hcy increases oxidative stress include inactivation of the antioxidant enzymes GPx-1 (230) or HMox (231), uncoupling of NOS (232), NF-κB- dependent activation of NOX (226) and inhibition of the Zn-binding capacity of metallothionein (229). Additionally, Hcy promotes mitochondrial dysfunction associated with increased global oxidative stress in cardiomyocytes (233, 234) and in the CNS Hcy induces a specific increase in mitochondrial oxidative stress (235).
The Cbl cofactors during the catalytic cycle are sensitive to oxidation, and both
MS and MMM are inactivated by ROS (236, 237). In the human endothelium, Hcy metabolism depends exclusively on Cbl-dependent MS (120). Impaired MS activity due to Cbl deficiency results in elevated Hcy which causes oxidative stress, thus further hindering Cbl metabolism.
Cbl deficiency is a common and significant public health problem, particularly amongst the elderly (114). Since the introduction of the folic acid fortification in the US diet, Cbl deficiency ranks above folate deficiency as the primary modifiable cause of hyperhomocysteinemia (238).
We hypothesized that L-Hcy could induce endothelial cell dysfunction through increasing ROS in human aortic endothelial cells (HAEC), and that Cbl can inhibit this dysfunction by reducing ROS levels.
The Specific Aims of this chapter are:
• To mimic a state of hyperhomocysteinemia in primary endothelial cells
and to determine if ROS are elevated in response to Hcy.
92
• To study the effect of L-Hcy and ROS production on cellular dysfunction
in HAEC.
• To determine whether pre-treatment with CNCbl can prevent the L-Hcy-
induced increase in ROS levels, and cellular dysfunction.
4.2 Experimental Section
Synthesis of L- and D-Hcy. L- or D-Homocysteine thiolactone (20 mg; 130 μmol) was dissolved in NaOH (5 N, 200 μl) and incubated at 37 ºC for 10 min. The solution was chilled and neutralized with HCl (5 N, 200 μl). PBS was added to a total volume of 1 ml and the solution bubbled with N2 for 10 min.(229) The yield was typically > 95%,
determined by quantifying the reduced thiol groups by the Ellman’s assay (199).
Cell culture. Primary HAEC were cultured as described in Chapter 3. Cells were
seeded onto 96- or 6-well plates at a density of 12,000-20,000 cells/cm2 and used for
experiments up to passage 6.
Assessing ROS production. Cells with or without CNCbl pre-treatment (500 pM -
10 μΜ) were incubated with L-Hcy (100 or 150 μM), H2O2 (50 - 200 μΜ), rotenone (5
μΜ), or with culture medium alone. To assess general ROS production, cells were
incubated with 3 μΜ dichlorofluorescein acetate (DCFA) for the duration of the L-Hcy or
- H2O2 treatment. For the assessment of O2• production, cells were incubated with 5 μM
DHE, or 5 μΜ MitoSOX for 1 h at the end of the L-Hcy, paraquat, or rotenone treatment.
Fluorescence was measured in a plate reader (DCF: λex/em = 420/520; hydroxyethidium:
λex/em = 520/605 nm; MitoSOX: λex/em = 510/580 nm).
93
Cell viability. Cell viability was assessed with trypan blue staining or with the
MTT assay. For the MTT assay cells were incubated with thiazolyl blue tetrazolium
(0.4 mg/ml) in M199 for 3 h at 37 ºC. The formazan crystals were resuspended in dimethylsulfoxide. Mitochondrial-dependent tetrazolium reduction to formazan was measured by reading optical density at 540 nm.
Cbl protection against L-Hcy or rotenone. Pre-confluent HAEC were incubated for 24 h in the absence or presence of CNCbl (500 pM – 10 μΜ) prior to adding L-Hcy
(100 or 150 μΜ), or rotenone (5 μΜ). For some experiments apocynin (0.1 mM) was added 30 min before adding L-Hcy. Cells were incubated for 24 h with L-Hcy, H2O2, or for 1 h with rotenone. ROS and cell viability were assessed as described above.
DNA and Cbl quantification. Confluent cells were harvested in lysis buffer (50 mM Tris pH 7.4, 0.5 % Triton X-100) and DNA quantified using the CyQuant cell proliferation kit (Roche). Cbl was quantified using the SimulTRAC Radioassay for vitamin B12 and folate by MP Biomedicals (Orangeburg, NY) according to the
manufacturer’s specifications.
Apoptosis measurements. To detect apoptotic cell death, cells were seeded onto 6- well plates and pre-treated with or without CNCbl (10 – 100 nM) for 24 h. Cells were washed, then incubated in the absence or presence of L-Hcy for 18 h. Apoptosis was assessed using the Cell Death Detection ELISA (Roche) according to the manufacturer’s specifications.
General solution preparation. Thiol solutions were prepared immediately before use and the concentrations were determined by the Ellman’s method (199). A fresh
94
solution of H2O2 was prepared before experiments and the concentration determined
-1 spectrophotometrically (ε240nm = 43.6 M.cm ) (239). The concentration of the stock
-1 solution of CNCbl was determined by the dicyanocobalamin test (ε368nm=30.4 mM.cm )
as described in Chapter 2 (197).
Statistics. All experiments were carried out using at least three separate cell
clones. Results are expressed as mean ± SEM. Statistical comparisons were carried out
using ANOVA with the Bonferroni post hoc test.
4.3 Results
4.3.1 Cobalamin Protection against homocysteine-induced Increase in ROS
Treatment of HAEC with varying concentrations of L-Hcy induced a
concentration-dependent increase in ROS detected by increasing dichlorofluorescein
(DCF) fluorescence, a general probe for ROS. L-Hcy (100 µM) elicited a significant
increase in ROS compared to control (Figure 4.1), consistent with previous studies (229).
To verify that the ROS increase was a L-Hcy-specific effect, HAEC were incubated for
48 h with 0.1 mM of a range of thiols (glutathione, L-cysteine, D-Hcy, L-Hcy, and β-
mercaptoethanol (βME)). Only L-Hcy elicited a significant increase in DCF fluorescence
(Figure 4.2A), concomitant with a significant decrease in cell viability as measured using
the MTT assay (Figure 4.2B).
Prior to determining the effect of vitamin B12 (CNCbl) on L-Hcy-dependent ROS
levels, the concentration and time-dependent uptake of CNCbl by HAEC was assessed.
Increasing the CNCbl in the medium (0.1 – 10 μΜ) led to higher intracellular Cbl levels
95
* 2.4 1.6 * 1.5 * 1.4 1.3 1.2 1.1 ROS fold increase 1.0 0 100 200 300 [Hcy] (μM)
Figure 4.1. L-Hcy-induced ROS increase. Pre-confluent HAEC were incubated with increasing concentrations of L-Hcy in the presence of 3 μM DCFA. After 48 h ROS production was assessed fluorometrically. *p <0.05 compared to control. Positive control exposed to 200 μΜ H2O2 (♦). Data are expressed as mean ± SEM; N = 3.
96
A B
130 1.6 * 120
1.2 110
100 0.8 90 * 0.4 80 cell viability (%) 70
ROS production (fold increase) 0.0 0 Ctl GSH L-Cys βME D-Hcy L-Hcy Ctl GSH L-Cys βME D-Hcy L-Hcy Thiol 0.1 mM Thiol 0.1 mM
Figure 4.2. L-Hcy specificity for eliciting ROS. Pre-confluent HAEC were incubated with varying thiols (0.1 mM) for 48 h. A. ROS production 48 h post treatment. B. Cell viability after 48 h. *p <0.05 compared to control untreated cells. Data are expressed as mean ± SEM, N = 3.
97
after 24 h (Figure 4.3). Incubating HAEC with CNCbl (0.2 nM) induced a time dependent increase in intracellular CNCbl as described in the previous chapter (Figure
3.3). A 24 h incubation time with CNCbl was selected as an appropriate time for all subsequent experiments. Exposing HAEC to L-Hcy (150 µM) over 48 h induced a 1.25- fold increase in DCF fluorescence (Figure 4.4A) that correlated with a ~ 25% decrease in cell viability (Figure 4.4B). To assess the effects of CNCbl on the L-Hcy-dependent ROS production and the L-Hcy-dependent decrease in cell viability, we pre-incubated HAEC with increasing concentrations of CNCbl for 24 h prior to treating the cells with L-Hcy
(150 μM). To ensure that extracellular Cbl was not responsible for Cbl effects on ROS, cells were washed after Cbl treatment and medium replaced prior to further treatments.
Pre-incubation of HAEC with CNCbl prevented the L-Hcy-dependent increase in ROS and decrease in cell viability in a concentration-dependent fashion (Figure 4.4). 10 nM
CNCbl completely inhibited the L-Hcy-dependent ROS increase (p < 0.05), whereas 50 nM of CNCbl was necessary to completely inhibit the L-Hcy-dependent decrease in cell viability. L-Hcy (150 μΜ) treatment of cells over a 24 h period resulted in a ~20% decrease in cell viability; hence, subsequent experiments were conducted using a 24 h
Hcy treatment protocol, unless otherwise stated.
4.3.2 Subcellular localization and cobalamin protection against homocysteine-
•- induced O2 increase
- It has been reported that L-Hcy treatment increases intracellular levels of O2•
- (240, 241). To determine if O2• is indeed an important ROS generated in our system,
- O2• levels upon exposure to L-Hcy were measured using hydroxyethidium fluorescence
98
50
40
30 g DNA g DNA μ μ / 12 20 fmol B
fmol Cbl/ 10
0 0 0.1 1.0 10
[CNCbl] (μM)
Figure 4.3. Effect of exogenous CNCbl on intracellular Cbl content. Pre-confluent
HAEC were incubated with or without varying concentrations of CNCbl for 24 h. The medium was then removed, the cells washed with PBS 3 times and harvested. The intracellular Cbl content was determined by the SimulTRAC radioassay. Data represents mean ± SEM; N = 3.
99
A 1.30 *
1.25
1.20
1.15
1.10 # ROS fold increase fold ROS 1.05
1.00 0.5 0.8 1.0 10 50 100 500 1000 [CNCbl] (nM) L-Hcy 150 μM
# B 100
90 * * 80 *
70 Cell Viability (%)
0 Ctl 0.5 0.8 1.0 10 50 100 500 1000 [CNCbl] (nM) L-Hcy 150 μM
Figure 4.4. Cbl protection against L-Hcy-induced oxidative stress. Pre-confluent
HAEC were incubated with increasing concentrations of CNCbl for 24 h prior to adding
150 μM L-Hcy. A. ROS fold-increase after 48 h. B. Cell viability after 48 h – MTT assay. Data are expressed as mean ± SEM; N = 7; *p<0.05 with respect to control;
#p<0.05 with respect to L-Hcy-treated cells not exposed to CNCbl.
100
•- as a O2 specific probe. Incubation of HAEC with L-Hcy (150 μΜ) for 24 h induced a
1.6-fold increase in hydroxyethidium fluorescence (Figure 4.5). This increase was
completely inhibited by pre-incubation of the cells with 10 nM CNCbl (p <0.05) or the
antioxidant apocynin (0.1 mM).
- To investigate the subcellular localization of the L-Hcy-induced increase in O2• ,
- cells were assayed with mitoSOX, a mitochondrial specific O2• probe. Incubation of
HAEC with L-Hcy (150 μM) for 24 h elicited a moderate but significant increase in
mitoSOX fluorescence, which was completely inhibited by preincubation of the cells
with CNCbl (50 nM) (Figure 4.6). As a control the effect of rotenone on mitochondrial
- O2• production was assessed. Rotenone inhibits the electron transport chain at the level
of Complex I, which facilitates the accumulation of reduced one electron transporters and
•- thus promotes the formation of O2 . Treatment of HAEC with rotenone (5 μM) induced
•- an increase in mitochondrial O2 which was partially reduced significantly by pre-
incubation with CNCbl (Figure 4.7).
4.3.3 Cobalamin protection against Homocysteine-induced Cell Death
Exposing HAEC to L-Hcy (150 μΜ) for 24 h caused a significant decrease in cell
viability as measured by MTT (Figure 4.7A). Since the MTT assay is a measure of
mitochondrial function and metabolic activity, cell death was directly assessed by trypan
blue staining, which corresponded with the MTT results (Figure 4.7B). Finally, to
characterize the L-Hcy induced cell death, apoptosis was assessed by measuring cytosolic
fragmented DNA with an ELISA-based cell death assay (Roche). HAEC showed a
101
Figure 4.5. Cbl protection against L-Hcy induced superoxide production. Pre- confluent HAEC were incubated in the absence or in the presence of 10 or 50 nM CNCbl for 24 h. Cells were washed with PBS, prior to incubation with 150 μM L-Hcy with or without 0.1 mM apocynin. After 24 h, cells were incubated with 5 μM DHE for 1 h (A).
Hydroxyethidium fluorescence was measured with a fluorescent plate reader before imaging. * p < 0.05 with respect to untreated HAEC; # p < 0.05 with respect to L-Hcy- treated cells. Data are expressed as mean ± SEM; N = 6.
102
A control cells L-Hcy (150 μM) L-Hcy + 50 nM Cbl
L-Hcy + 100 μM AC L-Hcy + 100 μM AC L-Hcy + 50 nM Cbl + 100 μM AC
B
2.0
* 1.6
# 1.2
0.8
0.4 production (fold increase) production -
• 2
O 0.0 Ctl 10 50 10 50 CNCblB12 (nM) (nM) AC 0.1 mM L-Hcy 150 μM
103
no pre-treatment pre-treated with 50 nM CNCbl # *
# 1.4 *
1.2
MitoSOXfluorescence (fold increase) 1.0 L-Hcy 150 μM rotenone 5 μM
Figure 4.6. Subcellular localization of Hcy-induced oxidative stress. HAEC were incubated in the absence or in the presence of CNCbl (50 nM) for 24 h. HAEC were
- washed and incubated with L-Hcy (150 μM, 24 h) or rotenone (5 μΜ; 1 h). O2• was evaluated by measuring the mitoSOX fluorescence (λex/em = 510/580 nm). Data are
expressed as mean ± SEM; N = 4; * p<0.05 compared to control, # p<0.05.
104
A B
30 120 # * 25 # 100 20
* 15 80 10 cell viability (%) 60 5 % trypan blue uptake %
0 0 AC 0.1 mM 10 50 CNCbl (nM) 10 50 10 50 CNCbl (nM) AC 0.1 mM μ Hcy 150 μM Hcy 150 M
Figure 4.7. Cbl protection against L-Hcy-induced cell death. Pre-confluent HAEC
were incubated in the presence or absence of 10 or 50 nM CNCbl for 24 h prior to adding
150 μM L-Hcy for 24 h in the presence or absence of apocynin (0.1 mM). *p<0.05 with
respect to control; #p<0.05 with respect to L-Hcy-treated HAEC. A Cell viability by the
MTT assay. Data are expressed as mean ± SEM; N = 4 B Trypan blue uptake. Data are
expressed as mean ± SEM; N = 3.
105
significant increase in apoptotic cell death in response to L-Hcy (150 μΜ) for 18 h
(Figure 4.8). Since both CNCbl and apocynin prevented L-Hcy-induced decrease in cell viability as measured by the MTT assay (Figure 4.7A), and the increase in cell death, as measured by trypan blue staining (Figure 4.7B) or ELISA (Figure 4.8), these data suggest that CNCbl can provide protection against apoptotic death.
4.4 Discussion
It is well established that Hcy is an independent risk factor for cardiovascular disease. Mouse models have shown a causal relationship between hyperhomocystenemia and atherosclerosis (reviewed by Zhou et al. (219)). The mechanisms proposed for Hcy- dependent cell dysfunction include activation of pro-inflammatory factors, oxidative stress, endoplasmic reticulum stress leading to activation of the unfolded protein response, and smooth muscle cell proliferation (220). Recently, it was shown that Cbl affords protection against Hcy-induced oxidative stress in a cell model using Sk-Hep-1 cells (89). These cells of endothelial origin are derived from the ascetic fluid of a patient with adenocarcinoma of the liver (242). Cancer-derived cells are known to behave differently from primary cells. Hcy promotes endothelial dysfunction by acting on endothelial and smooth muscle cells. Therefore, it was of interest to assess the possible protective role of Cbl in a model of hyperohomocysteinemia more relevant to cardiovascular disease using HAEC.
Hcy induced an increase in ROS in HAEC as previously reported (229). Our initial experiments established that L-Hcy (150 μM) increased DCF fluorescence (a measure of general intracellular ROS) by 1.25 fold in HAEC. Remarkably, this increase
106
1.6 *
1.4
1.2 #
1.0
0.8
apoptosis (fold increase) (fold apoptosis 0.6
10 50 100 100 CNCbl (nM)
AC 0.1 mM Hcy (150 μM)
Figure 4.8. Cbl protection against L-Hcy-induced apoptosis. Pre-confluent HAEC were incubated in the presence or absence of varying concentrations of CNCbl for 24 h prior to exposing the cells to 150 μM L-Hcy for 18 h in the presence or absence of apocynin (0.1 mM). Shown also are corresponding data for apocynin and apocyanin +
CNCbl. Apoptotic DNA fragmentation was measured by ELISA. Data are expressed as mean ± SEM; N = 4; *p<0.05 with respect to control; #p<0.05 with respect to L-Hcy-
treated HAEC.
107
and the associated decrease in cell viability (MTT assay) were completely inhibited by pre-treating HAEC with CNCbl (10-50 nM) prior to exposing them to Hcy. Importantly, these Cbl concentrations are physiologically relevant since Cbl supplementation can achieve 10-7 M levels in plasma (243, 244).
Hcy also induced a 1.6-fold increase in hydroxyethidium fluorescence (a specific
- •- probe for O2• ) in HAEC demonstrating that, in this system O2 is a major ROS produced.
This is in accordance with previous reports suggesting that Hcy-induced increase in ROS
•- is attributable to an increase in O2 levels (240, 241). Once again, this increase and the
associated decrease in cell viability were completely prevented by pretreating HAEC
with CNCbl (10 nM) or the addition of the antioxidant apocynin.
Elevated Hcy has been shown to cause mitochondrial dysfunction (233-235). It
was of interest to see if Hcy increases mitochondrial oxidative stress in HAEC and
whether CNCbl can prevent this, given that Cbl is present in mitochondria (Cbl-
dependent MMM is a mitochondrial enzyme). Hcy increases mitochondrial
lipoperoxidation in rat brain (235) and it has been proposed to increase mitochondrial
oxidative stress in cardiac myocytes (234). L-Hcy was found to induce an increase in
- mitochondrial O2• levels in HAEC, as indicated by the increase in mitoSOX fluorescence. Pretreatment with CNCbl (50 nM) prevented this Hcy-induced increase.
Furthermore, CNCbl partially inhibited the rotenone-dependent increase in mitochondrial
- O2• ; hence Cbl has antioxidant actions both in the cytosol and the mitochondria. To our
knowledge this is the first report of subcellular localization of Hcy-induced oxidative
stress. In our studies, Cbl was able to specifically inhibit the Hcy-induced increase in
108
•- •- mitochondrial O2 . An increase in mitochondrial O2 was also induced by blocking the
electron transport chain with rotenone. Cbl inhibited the rotenone-dependent increase in
•- •- mitochondrial O2 levels. Therefore, the ability of Cbl to inhibit mitochondrial O2 production is independent of Hcy metabolism.
Mitochondrial dysfunction and mitochondrial oxidative stress are associated with several pathologies including neurodegenerative disease, psychiatric disease, cardiovascular disease, sepsis and aging (245-251). Moreover, mitochondria are the effector organelles of the apoptotic intrinsic pathway (252). The central role of mitochondria as source and target for oxidative stress has led to an increased interest in the development of mitochondrion-targeted antioxidants such as the quinone mitoQ (250,
251, 253, 254). Antioxidants of this type rely on a positively charged phosphonium moiety that leads to an accumulation in the mitochondria driven by the mitochondrial transmembrane potential (255). On the other hand, Cbl is naturally delivered into the mitochondrial matrix to act as a cofactor for mitochondrial MMM. The cofactor distribution in vascular endothelial cells shows that there is a higher concentration of
AdoCbl in the cell compared to MeCbl, suggesting a very effective mechanism for mitochondrial delivery. Importantly, our results support the possibility that Cbl can act as an effective mitochondrial antioxidant.
Finally, both CNCbl and apocynin prevent Hcy-induced cell death measured by the MTT assay and trypan blue staining. L-Hcy induced an increase in apoptosis, assayed by measuring fragmented cytosolic DNA. Apoptotic cell death is a hallmark of atherosclerotic lesions. Apoptotic death has been suggested to increase the risk of
109
atherosclerotic lesion rupture. Moreover, apoptosis enhances thrombogenicity by increasing the number of tissue factor-rich apoptotic cells within the atherosclerotic lesion (256, 257). L-Hcy induces apoptosis in human bone marrow stromal cells (258), human umbilical vein endothelial cells (223, 224, 226, 227), and endothelial progenitor cells (225). It has also been shown to inhibit growth (259) and reduce cell viability in
HAEC (260). Our results are in line with previous studies for other types of endothelial cells. However, to our knowledge this is the first study showing that L-Hcy induces apoptotic cell death in HAEC. L-Hcy-induced apoptosis in HAEC was prevented by apocynin or pretreating HAEC with CNCbl (50 nM). Therefore, the ability of Cbl to inhibit apoptosis is directly relevant to the pathophysiology of CVD.
To summarize, physiologically relevant concentrations of CNCbl, the common
B12 form in vitamin supplements, were shown to effectively protect against L-Hcy and
- increased intracellular levels of O2• , both in the cytosol and in the mitochondria.
Accordingly, ROS-induced endothelial dysfunction was also prevented by CNCbl
treatment. B12-dependent MS catalyzes the reaction of Hcy with MeTHF to generate Met
and THF; hence, this is one mechanism by which Cbl protects against Hcy-induced
oxidative stress at the cellular level. However a direct effect of Cbl on ROS levels,
independent of Hcy metabolism cannot be discarded. Cbls have been administered in
high doses to treat pernicious anaemia for decades and CNCbl is routinely included in
vitamin supplements with no known toxicity, even at high doses (261). Our results
support the hypothesis that Cbl possesses antioxidant properties and have important
110
implications both with respect to the high percentage of the elderly who are B12-deficient and in the treatment of chronic diseases associated with oxidative stress.
111
CHAPTER 5
Vitamin B12 and redox homeostasis: Cob(II)alamin reacts with superoxide at rates
approaching superoxide dismutase
5.1 Introduction
•- The ROS O2 is produced by membrane electron transport systems and oxidase
enzymes. It is an important signaling molecule involved in many of the cell’s
•- physiological processes. O2 activates the MAPK pathway in endothelial cells, stimulates
sympathetic outflow in the central nervous system (63), and its antimicrobial activity is
•- directly involved in host defense (262). However, if O2 overwhelms its primary
enzymatic defenses, SOD, elevated levels of pro-inflammatory cytokines, chemotactic
factors and adhesion molecules ensue, and •NO is depleted with concomitant formation of
ONOO- (62). Low SOD levels are associated with oxidative stress (61), and there is
currently considerable interest in developing therapeutics that mimic SOD (62).
Cbl has numerous effects on the immune and inflammatory responses, effects that
are difficult to ascribe solely to its known coenzyme activities (132). Recent studies have
shown that Cbl protects against oxidative stress in a cell model (89). Importantly, Cbl has
also been shown to have antioxidant properties in Hcy-independent systems (89, 91,
263). Vitamin supplements that contain cyanocobalamin (CNCbl, vitamin B12) among other vitamins decrease LDL oxidation in both healthy individuals and in patients with coronary artery disease (264). Cbl supplementation is also beneficial in treating many
111 112
inflammatory diseases and there is accumulating evidence that Cbl can provide protection in oxidative stress-associated pathologies (77, 132-135, 137, 265). Levels of the Cbl transport protein TC have also been shown to be elevated during inflammation (266-268) concomitant with NF-κB activation (137), which is induced by various stimuli, including
ROS. Taken together, these studies suggest a potential role for Cbl in the regulation of inflammatory processes (125, 137).
Given that a major intracellular form of Cbl, cob(II)alamin (Cbl(II)) (237) reacts rapidly with the radical nitric oxide (•NO) to form nitroxylcobalamin
8 -1 -1 8 -1 •- (k = 7.4 x 10 M s , Keq~1 x 10 M , 25 °C (84, 88)), and that O2 reacts rapidly with
•- other Co(II) macrocycles (269), we hypothesized that scavenging of O2 by Cbl(II) might
be an important mechanism by which Cbl modulates intracellular signal transduction and
•- protects against chronic inflammation. A kinetic study on the reaction between O2 and
Cbl(II) was therefore carried out in order to directly determine the rate of this reaction.
The biological relevance of the reaction was assessed by testing the ability of Cbl to
•- protect HAEC against elevated O2 levels induced by paraquat.
The Specific Aims of this study are:
•- • To study the kinetics of the reaction between Cbl(II) and O2 .
•- • To test the ability of CNCbl to protect against O2 -dependent damage in a
human primary vascular cell model.
113
5.2 Experimental Section
5.2.1 General Methods
Materials. Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was purchased from Oxis
International (Foster City, CA). XO and catalase were obtained from Calbiochem, EMD
Chemicals (Darmstadt, Germany) and bovine heart ferricytochrome c, H2O2, and
acetaldehyde were purchased from Sigma (St. Louis, MO). Aquacobalamin
(HOCbl•HCl) was supplied by Fluka (Buchs, Switzerland).
Instrumentation. UV/visible spectra were recorded on a Cary 5000
spectrophotometer equipped with a thermostated cell changer (25.0 ± 0.1 °C), operating
with WinUV Bio software (version 3.00). A MBRAUN Labmaster 130(1250/78) glove
box operating under argon atmosphere was used. pH measurements were made at room
temperature with an Orion Model 710A pH meter equipped with a Mettler-Toledo Inlab
421 electrode. The electrode was filled with 3 M KCl / saturated AgCl solution, pH 7.0
and standardized with standard BDH buffer solutions at pH 4.01 and 6.98.
Anaerobic solutions were degassed by at least three freeze-pump-thaw cycles
under argon using standard Schlenk techniques. Data were fitted using OriginPro 8.0
(OriginLab Coorporation).
Synthesis of cob(II)alamin. Aquacobalamin (47 mg, 34 μmol) was dissolved in
2.0 ml anaerobic water. An anaerobic aqueous solution of NaBH4 (217 mM) was
prepared inside the glovebox. 1.1 mol equiv. (37 μmol, 170 μL) NaBH4 was added to the
aquacobalamin solution and the reaction was quenched with anaerobic acetone (50 μl)
114
after 15 min. The total conversion to cob(II)alamin was confirmed by recording a
UV/visible spectrum of the solution in a cuvette for anaerobic measurements (λmax = 313,
475 nm) (270). The anaerobic solution of Cbl(II) was kept in the glovebox until transfer
to a gas-tight septum-capped vial for immediate use.
Determination of Cbl and Cu,Zn-SOD concentrations. The concentration of Cbl
solutions were determined using the dicyanocobalamin test as described in Chapter 2
-1 -1 (ε368nm = 30.4 mM cm ) (197) with an uncertainty of ± 5%. The concentration of SOD
-1 -1 was determined using its known extinction coefficient at 258 nm (ε258 = 10,300 M cm )
(271).
Superoxide production. The superoxide flux generated using the acetaldehyde/XO system was determined using a well established assay involving reduction of ferricytochrome c by superoxide. Specifically, varying amounts of xanthine oxidase (6.0 -
40 μg/ml) were added to a cuvette containing acetaldehyde (20 mM) and ferricytochrome c (50 μM) in aerobic phosphate buffer (10 mM, pH 7.4, 1 mM EDTA) and the initial rate of ferricytochrome c reduction determined spectrophotometrically at 550 nm (Δε550nm =
21 mM-1cm-1) (272). Figure 5.1 shows the relationship between enzyme concentration
•- and O2 flux. Under these conditions the spontaneous dismutation of superoxide is
•- negligible and the initial rate of ferricytochrome c reduction equals the O2 flux.
Determination of the molar extinction coefficients for Cbl(II) and
+ H2OCbl /HOCbl at 351 nm, pH 7.4. The absorbance at 351 nm of solutions of Cbl(II)
(5.00 to 1.00 x 102 μM) in anaerobic phosphate buffer (10 mM, pH 7.4) were recorded
115
16 14 )
-1 12 10 M.min μ 8
flux ( 6 -
• 2
O 4 2 0 010203040 [xanthine oxidase] (μg/ml)
•- •- Figure 5.1. O2 production by xanthine oxidase. Plot of O2 production rate vs
concentration of xanthine oxidase. Xanthine oxidase (6.0 – 40 μg/ml) was added to a
solution containing acetaldehyde (20 mM) and ferricytochrome c (50 μM) in phosphate buffer (10 mM, pH 7.4, 1 mM EDTA, 25.0 °C). The initial rate of ferricytochrome c
•- reduction was followed at 550 nm and the O2 flux calculated.
116
(25.0 °C). The solutions were then exposed to air for 24 h, allowing for total oxidation of
+ Cbl(II) to Cbl(III) (= H2OCbl /HOCbl) and the absorbance at 351 nm re-measured. The absorbance at 351 nm versus the Cbl concentration was plotted for Cbl(II) and Cbl(III) and the extinction coefficients for each species calculated from the slope (for Cbl(II)
-1 -1 -1 -1 -1 -1 ε351nm = 12.6 mM cm ; for Cbl(III) ε351nm = 27.2 mM cm ; Δε351nm = 14.6 mM cm ).
-1 -1 + A value of ε351nm = 26.5 mM cm for H2OCbl in water had been previously reported
(270).
5.2.2 Kinetic Measurements
Kinetics of oxidation of cob(II)alamin by superoxide. XO (6.0 - 32 μg/mL) was added to a cuvette containing acetaldehyde (20 mM) and catalase (1000 U/ml), in aerobic
10 mM phosphate buffer, pH 7.4, containing 50 mM NaCl and 1 mM EDTA, which had been pre-equilibrated to 25.0 °C. To initiate the reaction, an aliquot of anaerobic Cbl(II) solution in phosphate buffer (10 mM, pH 7.4) was added ~5 s after adding the XO using a gas-tight syringe. The initial rate of oxidation of Cbl(II) was followed at 351 nm
(formation of aquacobalamin) or 474 nm (disappearance of Cbl(II)) for ~30 s. In some experiments, spectra (300-600 nm) were also recorded to confirm that Cbl(II) is indeed completely converted to aquacobalamin.
The upper limit of the superoxide flux was set to 20% of the Cbl concentration
per minute (i.e., maximum of 10 μM min-1 for a Cbl concentration of 50 μM), in order to maintain less than 10% Cbl(II) consumption during the kinetic measurement (30 s)). Note that in the absence of acetaldehyde, XO does not induce an acceleration in Cbl(II)
116 117
oxidation above that of air-catalyzed oxidation, nor does acetaldehyde in the absence of
XO.
Initial rates of Cbl(II) oxidation (in units of min-1) were obtained from the slopes
of plots of change of absorbance versus time. These values were subsequently converted
-1 -3 to initial rates in units of μM.min by dividing the rates by Δε351nm = 14.6 x 10
µM-1.cm-1 (see above).
Competition kinetics with SOD. An aliquot of an anaerobic Cbl(II) solution (final
concentration 50.0 ± 2.5 μM) was added to a cuvette containing xanthine
•- oxidase/acetaldehyde (20 mM) (to achieve 10 μM/min O2 ) and 1000 U/ml catalase in
aerobic phosphate buffer (10 mM, pH 7.4 with 50 mM NaCl and 1 mM EDTA) at
25.0 ºC as described above. The same experiment was repeated in the presence of
increasing concentrations of Cu,Zn-SOD (2.0 - 88 μM), which was added to the buffer
solution prior to the addition of XO and Cbl(II). Note that the concentration of enzyme
•- •- that gave a flux of 10 μM/min O2 was ~33 μg/ml. However, the O2 flux was measured
before each experiment to account for inactivation of the enzyme and the concentration
•- of XO then adjusted as necessary to obtain the desired O2 flux.
To confirm that there was no direct reaction between Cbl(II) and SOD a control
experiment was performed. SOD (28 μM) was added to an anaerobic solution of Cbl(II)
(28 μM) in phosphate buffer 10 mM pH 7.4 with 50 mM NaCl and 1 mM EDTA. The reaction was followed at 351 nm. There was no absorbance change over a period of 5 min
(ΔA/min < 0.0001).
118
•- The rate of O2 -dependent Cbl(II) oxidation was obtained by measuring the initial slope at 351 nm or 474 nm for an identical reaction mixture in the absence of xanthine oxidase and SOD, and subtracting the initial slope of autoxidation (autoxidation351nm <
0.017 min-1). The initial rate at 351 nm was converted to initial rates in units of μM min-1
-3 -1 -1 by dividing the rates by Δε351nm = 14.6 x 10 µM cm (see above).
5.2.3 Intracellular Studies
Cell culture. HAEC were cultured as described in Chapter 3. For all experiments
cells were plated in 96-well at a cell density of 20,000 cells/cm2. Cells were used for
experiments up to passage 6.
•- Assessing the protective effect of CNCbl on paraquat-dependent O2 production
and cell viability. HAEC were incubated with or without CNCbl (100 nM) for 24 h. The
media was then removed and cells were washed with PBS to remove extracellular Cbl.
•- The cells were then incubated with paraquat (1.5 mM) for 24 h prior to assessing O2
•- production using a fluorescent O2 -detection assay. When SOD was used, the enzyme was added at a concentration of 3 μM at the same time as the paraquat. When apocynin was used, it was added at a concentration of 0.1 mM 1 h prior to the paraquat addition and kept in the medium until the fluorescence assay was performed. After 24 h, the
•- medium was removed, the cells incubated with the O2 -sensitive probe dihydroethidium
(5 μM) for 1 h and the fluorescence was read in a plate reader (λex = 480 nm, λem = 620
nm) as described in Chapter 4. Cell viability was assessed with the MTT assay as
described in Chapter 4.
119
5.3 Results
•- 5.3.1 In vitro Kinetic Studies on the Reaction between Cbl(II) and O2
•- •- -1 The reaction between Cbl(II) (33 μM) and O2 (O2 flux = 2.7 μM.min ) was followed by UV/visible spectrophotometry, recording a spectrum every 1.00 min. Upon
•- the addition of O2 produced by the XO /acetaldehyde system, Cbl(II) was cleanly
+ converted to aquacobalamin (H2OCbl /HOCbl, pKa ~ 7.8; λmax = 353, 413, 501, 530 nm
(44, 270)), with sharp isosbestics at 337, 375, 490, and 578 nm (Fig. 5.2). Assuming that
-1 -1 + the reaction goes to completion, since ε351nm = 27.2 mM cm for H2OCbl /HOCbl at pH
= 7.4, 25.0 °C, the expected final absorbance at the end of the reaction was 0.898. After
13 min the measured final absorbance at 351 nm was 0.900 (Figure 5.2A). This indicated
that Cbl(II) was therefore completely converted to Cbl(III) during the reaction.
•- Furthermore, the plot of initial rate of Cbl(II) oxidation vs O2 flux has a slope of 1.2 ±
•- 0.1 (Figure 5.2B), as expected if Cbl(II) reacts directly with O2 . This value slightly
above unity is probably due to the O2-dependent oxidation of Cbl(II).
•- + O2 is reduced to H2O2, which can also oxidize Cbl(II) to H2OCbl /HOCbl (273).
Indeed, adding 0.5 mole equivalents H2O2 to a solution of Cbl(II) resulted in the
+ formation of H2OCbl /HOCbl (Figure 5.3). Catalase (1000 U/ml) rapidly
disproportionates H2O2 to O2 and H2O, and the rate of Cbl(II) oxidation was decreased by
the addition of catalase as expected (Figure 5.4). Increasing the amount of catalase to
2000 U/ml did not cause any further decrease in the rate of oxidation; thus, all kinetic
experiments were carried out in the presence of 1000 U/ml catalase. H2O2 also reacts
+ with H2OCbl /HOCbl, albeit at a much slower rate (273); however, the addition of up to
120
A
1.0
0.8
0.6
0.4 Absorbance
0.2
0.0 300 350 400 450 500 550 λ (nm) B 14 )
-1 12 10 M.min
μ 8 6 4 2 oxidation rate ( rate oxidation 0
024681012- -1 O ∗ flux (μM.min ) 2
Figure 5.2. Cob(II)alamin oxidation by superoxide. A. UV/visible spectra recorded
•- •- every 1.00 min for the reaction between Cbl(II) (33 μM) and O2 (O2 flux = 2.7
•- μM/min) in phosphate buffer, 25.0 ºC. B. Initial rate of Cbl(II) oxidation versus O2 flux in the presence of 1000 U/ml catalase, in phosphate buffer, 25.0 ºC. The slope of the plot is 1.2 ± 0.1. Data are expressed as mean ± SD; N = 3.
121
0.42
0.41 351nm 0.40 Abs
0.39
0.0 0.2 0.4 0.6 0.8 1.0 Time (min)
Figure 5.3. Cob(II)alamin reacts with H2O2. Plot of absorbance at 351 nm versus time
for the reaction between Cbl(II) (20 μM) and H2O2 (10 μM) in 10 mM phosphate buffer
pH 7.4, at 25.0 ºC.
122
0.14
0.12
0.10
0.08 351nm
Abs 0.06
0.04
0.02
0.00 0.0 0.2 0.4 0.6 0.8 1.0 Time (min)
Figure 5.4. Catalase decreases the initial rate of Cbl(II) oxidation. Plot of absorbance
•- •- at 351 nm versus time for the reaction between Cbl(II) (20 μM) and O2 (O2 production
= 5 μM/min using the xanthine oxidase/acetaldehyde system) in 10 mM phosphate buffer, pH 7.4, at 25.0 ºC in the absence (black; slope = 0.102 min-1) or presence (grey;
slope = 0.062 min-1) of catalase (1000 U/ml).
123
+ 5 molar equivalents of H2O2 to H2OCbl /HOCbl did not induce any change in the
UV/visible spectrum in the time frame of the experiments (ΔAbs351nm < 0.001); hence this
reaction is unimportant.
+ The second-order rate constant for the oxidation of Cbl(II) to H2OCbl /HOCbl by
•- O2 was determined using a well established competition kinetics method. This method
has been validated by others by comparing rate constants obtained using a competition
approach with those obtained directly using pulse radiolysis methods (274). In this
method, the rate of the reaction was measured in the presence of varying concentrations
of a competitor, Cu,Zn-SOD:
•- + cob(II)alamin + O2 + 2H cob(III)alamin + H2O2
•- SOD + O2 products
In this model the rate at which Cbl(II) (= Cbl) is consumed is:
= .− CblOkV ][][ 0 Cbl 2 0 (5.1)
where V0 is the observed rate of Cbl(II) oxidation in the absence of SOD, kCbl is the
•- second-order rate constant for the reaction between O2 and Cbl(II), [Cbl] is the Cbl(II)
•- •- concentration and [O2 ]0 is the O2 concentration in the absence of a competitor (SOD).
In the presence of SOD:
= .− CblOkV ][][ SOD Cbl 2 SOD (5.2)
•- where VSOD is the observed rate of Cbl(II) oxidation in the presence of SOD, [O2 ]SOD is
•- the O2 concentration in the presence of the competitor.
Hence,
123 124
⎛ V ⎞ ⎛ []O.− ⎞ ⎜ 0 ⎟ =⎜ 2 0 ⎟ (5.3) ⎜ ⎟ ⎜ .− ⎟ ⎝VSOD ⎠ ⎝ []O2 SOD ⎠
•- •- Assuming a steady state concentration of [O2 ], the rate of production of O2 by
•- the XO/acetaldehyde system (J) equals the rate of O2 consumption.
Therefore, without a competitor:
= .− ][ CblOkJ Cbl 2 0 [] (5.4) and with SOD:
.− = 2 (][ CblSOD []− SOD SODkCblkOJ ])[ (5.5)
•- where kSOD is the rate constant for enzymatic dismutation of O2 to H2O2 + H2O for
9 -1 -1 Cu,Zn-SOD (2 x 10 M s (64)), and kCbl is the rate constant for the oxidation of Cbl(II)
•- by O2 .
•- Solving for [O2 ] and substituting into equation (5.3) gives:
V k 0 1 =− SOD comp][ (5.6) V Cblk ][ SOD Cbl
•- By measuring the rate of Cbl(II) oxidation by a flux of O2 produced by the
XO/acetaldehyde system in the presence of increasing concentrations of a competitor, it is possible to determine the second-order rate constant kCbl.
•- The initial rate of oxidation of Cbl(II) by O2 was inhibited by Cu,Zn-SOD in a concentration dependent fashion (Fig. 5.5A and 5.5C). The rate constant for the reaction
•- between Cbl(II) and O2 was calculated using equation (5.6), assuming a steady state
•- concentration of O2 . The scatter of the rate data (ΔAbs/min) was similar for all experiments (~0.01 min-1). However, from Fig. 5.5B and 5.5D it is clear that increased
125
Figure 5.5. Competition kinetics with Cu,Zn-SOD. A. Plot of absorbance at 351 nm
•- (A) or 474 nm (C) (dashed line) versus time for the oxidation of Cbl(II) (50 μM) by O2
•- (O2 flux = 10 μM/min; 1000 U/ml catalase, 1 mM EDTA, 50 mM NaCl, 10 mM
phosphate buffer, pH 7.4, 25.0 ºC). The experiment was repeated with increasing SOD
(2-88 μM, solid lines; arrow indicates increasing SOD concentration). The rate of Cbl(II)
autoxidation is also shown (dotted line). B: Plot of V0/VSOD - 1 vs. [SOD] at 351 nm. The
8 -1 -1 best linear fit to eqn (5.6) gave kCbl = (6.8 ± 0.8) x 10 M s . D: Corresponding plot of
V0/VSOD - 1 vs. [SOD] at 474 nm. The best linear fit resulted in a slope of 0.049 ± 0.003
-1 8 -1 -1 μM , corresponding to kCbl = (8 ± 1) x 10 M s .
126
A B
0.20 7
0.16 6
5 0.12
)-1 4 351nm SOD
0.08 /V 3 0 Abs (V Δ 2 0.04 1
0.00 0 0.0 0.2 0.4 0.6 0.8 1.0 0 20406080100 Time (min) [SOD] (μM)
C D 7 0.65 6
5 0.64 4 )-1 474nm 0.63 SOD 3 /V 0 Abs (V Δ 2 0.62 1
0 0.0 0.1 0.2 0.3 0.4 0.5 0 20 40 60 80 100 Time (min) [SOD] (μM)
127
scatter in the data occurs at high SOD concentrations as expected, given that the plots use the ratio V0/VSOD, which has a larger relative error at smaller VSOD values (i.e. high
8 -1 -1 [SOD]). The best fit of the data at 351 nm gave kCbl = (6.8 ± 0.8) x 10 M s (Fig. 5.5B).
8 -1 -1 Similar experiments at 474 nm gave kCbl = (8 ± 1) x 10 M s (Fig. 5.5D). The smaller
ΔAbs at 474 nm resulted in a less reliable value of kCbl.
Importantly, since XO requires oxygen for its activity, the experiments were conducted in aerobic buffer. However, Cbl(II) undergoes slow disproportionation to
+ cob(I)alamin (Cbl(I)) and Cbl(III) (= H2OCbl /HOCbl) in air, followed by rapid oxidation of Cbl(I) to Cbl(III) (208, 275). It was therefore necessary to correct the data for autoxidation of Cbl(II). Cbl(II) autoxidation was measured in the absence of XO and the rate of Cbl(II) autoxidation subtracted from the V0 and VSOD data (dotted line in Fig.
+ •- 5.5A and 5.5C). The contribution of the reaction between H2OCbl /HOCbl and O2 to form superoxocobalamin (this species has been characterized at low temperature) has previously been shown to be negligible on the timescale of our experiments (276, 277).
The validity of our system was further tested by simulations carried out using the program Gepasi 3.30 (available from www.gepasi.org) (278, 279) and the following model:
v - O2 - - + k1 O2 + O2 {+ 2H } H2O2 + O2 - + k2 Cbl(II) +O2 {+ 2H } Cbl(III) +H2O2 - + k3 O2 + SOD{+ H } 1/2H2O2 + 1/2O2
127 128
The values used were: [Cbl(II)] = 20, 33 or 50 μM; [SOD] was varied between 0 and 88 μM; the initial concentrations of O2, H2O2, and Cbl(III) were set to 0; all the concentrations were allowed to vary except the SOD concentration which was fixed in
-1 5 -1 -1 8 -1 -1 9 -1 -1 each run; v = 10 μM.min , k1 = 2 x 10 M s , k2 = 7 x 10 M s and k3 = 2 x 10 M s
(k1 and k3 from reference 15, k2 from this work). Under these conditions, a steady state
•- [O2 ] was reached in less than 5 ms. The Cbl(II) oxidation rates (inital [Cbl(II)] = 50 μM) measured as appearance of Cbl(III) obtained from the simulations were in excellent agreement with our experimental data (Table 5.1).
Ferricytochrome c and the MnIII SOD mimetic MnIII meso-tetrakis(o-N- ethylpyridinium-2’-yl)porphyrin (MnTEPyp) were also tested for their suitability as competitors; however, determination of an accurate rate constant for the oxidation of
•- Cbl(II) by O2 was not possible using either complex. MnTEPyp catalyzes the
•- 7 -1 -1 dismutation of O2 with a second-order rate constant of (5.8 ± 1.2) x 10 M s (274).
•- Given that Cbl(II) reacts approximately one order of magnitude faster with O2 , high concentrations of the Mn complex are required; however the large extinction coefficient of the porphyrin made it impossible to accurately measure the rate of oxidation of Cbl(II).
A similar problem was encountered using ferricytochrome c as a competitor as the
•- reaction of O2 with ferricytochrome c is over three orders of magnitude slower than with
Cbl(II) (k = 2.6 x 105 M-1 s-1) (272) Moreover, there is a direct reaction between ferricytochrome c and Cbl(II) (Figure 5.6).
There is currently considerable interest in discovery of compounds that efficiently
•- scavenge O2 , given their effectiveness in ameliorating conditions associated with
129
•- Table 5.1. Experimental and simulated rates of Cbl(II) oxidation by O2 in the presence of SOD.
[SOD] μM Cbl(II) oxidation rate (μM/min)
Experimental Simulated
mean ± SD
0.0 11.5 ± 1.2 10.2
1.9 8.6 ± 0.6 9.1
3.7 7.8 ± 0.4 8.3
9.4 6.9 ± 0.4 6.5
16.9 5.9 ± 0.4 5.2
26.2 4.5 ± 0.2 4.0
33.7 3.9 ± 0.4 3.5
43.1 2.8 ± 0.4 2.9
88.0 2.0 ± 0.5 1.7
130
1.39
1.38
1.37
373nm
Abs 1.36
1.35
1.34
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Time (min)
Figure 5.6. Cbl(II) reacts directly with ferricytochrome c. Plot of absorbance at 373 nm versus time directly after the addition of 1 mol equiv. ferricytochrome c to Cbl(II) (20
μM) in phosphate buffer, pH 7.4 containing 1 mM EDTA, 25.0 ºC. The reaction was followed at 373 nm since this is an isosbestic wavelength for the conversion of Cbl(II) to
+ H2OCbl /HOCbl.
131
•- oxidative stress (62). Table 5.2 summarizes rate constants for the reaction between O2
•- and various O2 scavengers, including Cbl(II). To our knowledge, only one known SOD
•- mimetic, M40401, reacts faster with O2 than Cbl(II) (62).
•- 5.3.2 Cbl Protects Against Intracellular O2
In our in vitro studies, Cbl(II) was found to be a very efficient stoichiometric
•- scavenger of O2 . The physiological relevance of this finding is especially important since cells have the ability to re-reduce Cbl(III) to Cbl(II) (33); thus, the reaction between
•- Cbl and O2 is potentially catalytic in cell systems. In addition to reacting rapidly with
•- O2 , Cbl has the significant advantage of being non-toxic even at high doses (261), which
•- makes it a very good candidate as a O2 scavenger in vivo.
•- Although Cbl was shown to effectively inhibit Hcy-induced O2 levels measured by hydroxyethidium fluorescence (Chapter 4), it is not clear whether this is due to Cbl acting as an intracellular antioxidant or an increase in Cbl-dependent MS activity leading
•- to increased Hcy clearance (280). To test whether Cbl can specifically scavenge O2 in
•- HAEC, cells were challenged with paraquat (1.5 mM, 24 h), a well established O2 -
•- producing chemical. Paraquat elevates intracellular levels of O2 by redox cycling with
•- cellular diaphorases (281). Paraquat exposure elicited a 1.5-fold increase in O2 levels measured by hydroxyethidium fluorescence (Figure 5.7). The paraquat-induced increase in oxidative stress correlated with a 30% decrease in cell viability (Figure 5.7).
Pretreating HAEC with 10 nM CNCbl prior to exposing cells to paraquat prevented the increase in hydroxyethidium fluorescence and pre-incubation with 100 nM CNCbl
131 132
•- Table 5.2. Second-order rate constants for the reaction of various O2 scavengers
•- with O2 .
•- -1 -1 O2 scavenger k (M s )
TEMPOL 3.4 x 105 [a]
FeIII tris[N-(2-pyridylmethyl)-2-aminoethyl]amine 2.2 x106 [b]
~ 3 x 106 [c]
FeIII tetrakis(4-N-methylpyridyl)porphine 3 x 107 [d]
FeII tetrakis-N,N,N’,N’(2-pyridylmethyl)ethylendiamine ~ 3 x 107 [c]
MnIII meso-tetrakis(ortho-N-ethylpyridinium-2’-yl) porphyrin 5.8 x 107 [e]
MnIII meso-tetrakis(ortho-N-methylpyridinium-2’-yl) porphyrin 6.0 x 107 [e]
MnIII 5,10,15,20-tetrakis[N-methyl-N'-(2-methoxyethyl) imidazolium-2- 9.5 x 107 [f] yl]porphyrin
MnIII 5,10,15,20-tetrakis[N-(2-methoxyethyl) pyridinium-2-yl]porphyrin 1.1 x 108 [f]
SC-55858 1.2 x108 [g]
MnIII tetrakis(N-(1-(2-(2-(2-methoxyethoxy)ethoxy) ethyl)pyridinium -2- 1.3 x108 [h] yl)porphyrin
MnIII tetrakis(N,N'-di(1-(2-(2-(2-methoxyethoxy) ethoxy)ethyl)imidazolium-2- 3.5 x 108 [h] yl)porphyrin
cob(II)alamin 7 x 108 [i]
M40401 1.6 x 109 [g]
[a] Reference (282). [b] Reference (283). [c] Calculated from data in reference (284). [d] Reference (285). [e] Reference (274). [f] Reference (286). [g] Reference (62). [h] Reference (287). [i] This work.
133
Figure 5.7. Effect of Cbl on paraquat-induced cell death. HAEC were incubated in the absence or in the presence of varying concentrations of CNCbl for 24 h. Paraquat (1.5 mmol/L) was then added to cells for 24 h and DHE (5 µmol/L) was added for the final 1 h of treatment. Also shown are the effects of SOD (3 µmol/L) and apocynin (AC, 0.1
- - mmol/L) on the paraquat-induced increase in O2• and cell death. (A) O2• measured as
hydroxyethidium fluorescence (λex/em = 520/605 nm) compared to untreated cells (B) Cell
viability was assessed with the MTT assay. Data expressed as ± SEM; N = 3; * p<0.05
compared to control, # p<0.05 compared to paraquat.
134
A * 1.5 #
1.0
0.5 production (fold increase) production - • 2 0.0 O Ctl 10 50 100
CNCblB12 (nM)(nM) SOD AC Paraquat 1.5 mM B # 100
80 * 60
40
cell viability (%) 20
0 Ctl 10 50 100
CNCblB12 (nM)(nM ) SOD AC Paraquat 1.5 mM
135
prevented the accompanying decrease in cell viability. The same effect was observed with SOD (3 μM) itself or with the antioxidant apocynin (0.1 mM) (Figure 5.7).
5.4 Discussion
The in vitro kinetic studies show that Cbl(II), an important intracellular form of
•- 8 Cbl, reacts rapidly with O2 at rates approaching those of SOD itself (7 x 10 versus 2 x
9 -1 -1 •- 10 M s , respectively, (64)). This suggests that direct scavenging of O2 is an important molecular mechanism by which Cbl can modulate intracellular signal transduction and regulate processes associated with oxidative stress, such as chronic inflammation. The calculated rate constant is in the same order of magnitude as those reported for the reaction of Cbl(II) and •NO (88) and for the reaction of other Co(II)
•- macrocycles with O2 (269). The excellent agreement of the experimental results with the simulated data further supports the model. This result suggest that direct scavenging of
•- O2 is a plausible molecular mechanism by which Cbl modulates inflammation.
•- Importantly, upon reacting with O2 , Cbl(II) is oxidized to Cbl(III). For the reaction to be catalytic, Cbl(III) must be reduced back to Cbl(II). Specific reductases exist to enable cofactor biosynthesis, reactivate inactive Cbl-dependent enzymes (22, 36, 236) or even to protect the enzyme from oxidative inactivation (237). Indeed, when in turnover, ~80% of the Cbl associated with MMM is in the Cbl(II) form (237).
•- The physiological relevance of the scavenging of intracellular O2 by Cbl(II) was
•- probed by studying the effect of Cbl supplementation in HAEC challenged with the O2 - generating agent paraquat. Pre-treating HAEC with CNCbl was found to provide
•- excellent protection against O2 in this cell system. CNCbl (10 nM) prevented the 135 136
•- paraquat-associated increase in intracellular O2 levels and the corresponding decrease in
•- cell viability. These results suggest that direct scavenging of O2 by Cbl independent of
Hcy metabolism is an important mechanism by which Cbl protects against intracellular
oxidative stress. Our results support the hypothesis that Cbl can act as a second line of
•- defense when O2 production overwhelms the SOD protection system. This perhaps
accounts for significantly increased oxidative damage markers in patients with inherited
disorders of intracellular Cbl metabolism (288).
There is accumulating evidence that Cbl has other roles in biology in addition to
its cofactor role in the two mammalian B12-dependent enzyme reactions (reviewed by
Solomon) (125). Importantly, Cbl supplementation can be beneficial in treating a range of
inflammatory and viral based diseases (77, 132-135, 137, 265). Furthermore Cbl modulates the immune response (289, 290) and Cbl deficiency is associated with increased expression of the pro-inflammatory cytokine TNF-α and nerve growth factor,
and with reduced expression of interleukin-6 and epidermal growth factor. Cbl therapy
normalizes levels of TNF-α and epidermal growth factor in Cbl deficient patients (289).
However, the mechanism(s) by which Cbl achieves these effects are currently unclear.
Finally, it was recently suggested that Cbl inhibits NOS by blocking oxygen
binding to the active site of the enzyme (291). Weinberg et al. have suggested that
HOCbl and other corrinoids are capable of regulating •NO production by means of this
inhibitory effect, independent of their ability to scavenge •NO. This could partially
account for Cbl’s capacity to modulate inflammation. However, the concentrations
needed to exert this inhibition are much higher than those obtainable using B12
137
supplementation. Modulation of TNF-α production, on the other hand, can explain Cbl-
dependent inhibition of •NO production. This pro-inflammatory cytokine induces iNOS
in the CNS, causing an increase in •NO production (292), and Cbl successfully returns
TNF-α to physiological levels (289).
Given that B12 is non-toxic even at high doses and that a significant proportion of the elderly are B12 deficient, our results provide a compelling argument for further
exploration of the pharmacological benefits of B12 in the treatment and prevention of
diseases associated with chronic inflammation and aging.
138
CHAPTER 6
Summary and Future Directions
6.1 Summary
This dissertation explores the relationship between redox status and Cbl metabolism in the vascular endothelium. Vitamin B12 is a pivotal cofactor in the
cardiovascular system. Cardiovascular cells depend exclusively on MeCbl-dependent MS
to metabolize Hcy, elevated levels of which are an independent risk for CVD; hence Cbl
status has a direct impact on cardiovascular health. All cells in the body require B12, and in order to reach the tissues, B12 must be transported through the vasculature. Endothelial cells therefore play a key role in Cbl homeostasis. Oxidative stress is the common denominator at the center of CVD. Therefore, an understanding of the effect of oxidative stress on Cbl metabolism, and the effect of Cbl status on the antioxidant capacity of the cell, is crucial to understand the relationship between Cbl status and endothelial pathophysiology.
We have shown for the first time that the GSH status of the cell affects the Cbl
profile in human vascular endothelial cells when CNCbl is used as the Cbl source. Upon
GSH depletion, the total intracellular Cbl concentration is lowered, the relative amounts
of the cofactors is altered and there is no detectable GSCbl, unlike in non GSH-depleted
endothelium. Therefore the GSH status of the cell and/or the redox of the cell status
profoundly affects intracellular Cbl processing in the vascular endothelium.
138 139
We have demonstrated that physiologically relevant concentrations of CNCbl
•- effectively protect against Hcy- and paraquat-induced increases in O2 levels and
associated cell death. The antioxidant effect of Cbl appears to be both cytosolic and
•- mitochondrial and the ability of Cbl to reduce O2 levels is independent of Hcy
metabolism.
•- We have also demonstrated that Cbl(II) reacts with O2 at a rate approaching that
•- of SOD itself. This supports the hypothesis that Cbl directly scavenges O2 ; hence,
•- accounting for its protective effect aginst O2 -mediated oxidative stress in HAEC.
Our results highlight the interrelationship between redox status of the cell and Cbl
metabolism in the vascular endothelium and strongly suggest the need for further studies
on the therapeutic potential of Cbl for the treatment of CVD and other pathologies
associated with oxidative stress.
6.2 Future Directions
6.2.1 Glutathionylcobalamin and Cobalamin Processing.
It was shown that GSH depletion leads to an impairment in the cell’s ability to
process CNCbl and synthesize the Cbl cofactors. The MMACHC protein is essential for
reductive decyanation of CNCbl, an early step in Cbl processing, and GSH is required for
MMACHC-catalyzed reductive de-alkylation of alkylcobalamins. Further studies are
required to elucidate the mechanism(s) by which low GSH levels lead to impaired Cbl
processing. It is possible that impairment of Cbl processing due to GSH depletion could
be overcome with a Cbl source other than CNCbl for cofactor biosynthesis. If reduced
140
+ intracellular GSH levels primarily affects β-axial ligand removal, H2OCbl and/or
RSCbls are expected to be more effective Cbl sources in a GSH-depleted model. The
direct involvement of GSCbl in Cbl cofactor synthesis remains to be explored. Using
GSCbl as the source would indirectly assess the possible involvement of this metabolite
as an intermediate in cofactor biosynthesis.
The effect of other pro-oxidant stimuli on Cbl processing remains unknown. The
Cbl profile in cells challenged with different ROS or RNS could be of interest in order to
further probe the link between oxidative stress and Cbl metabolism.
•- 6.2.2 Cobalamin as a direct O2 scavenger
In Chapters 4 and 5 it is shown that CNCbl supplementation protects the vascular
endothelium against Hcy- and paraquat-induced oxidative stress. In vitro kinetic studies
•- support Cbl being an intracellular O2 scavenger. Further studies on the benefits of Cbl
supplementation in systems deficient in SOD would provide direct evidence for or
against this mechanism. We hypothesize that when the SOD defenses are overwhelmed
•- by elevated O2 levels, Cbl acts as a second line of defense. This could be further probed
•- using SOD knockdown cells. O2 production and cell viability could be measured in
siRNA SOD1 and SOD2 knockdown cells (293, 294) in the presence or absence of Cbl to
assess if Cbl is capable of compensating the lack of dismutase activity.
•- The hypothesis of direct scavenging of O2 by Cbl could be further investigated in
an animal model using SOD knockout mice (295). Homozygous SOD2 knockout mice die within the first three weeks of age, have decreased body size, exhibit progressive limb
weakness, anemia, neurodegeneration, abnormal mitochondrial physiology, decreased
141
adipose tissue mass and skeletal muscle mass, impaired myelopoiesis, abnormal hepatocyte morphology and cardiomyopathy (295). The effect of Cbl supplementation on these various neurological, skeletal, hematological and cardiovascular parameters could be studied. The ability of Cbl to improve or normalize any affected parameters would indicate not only that Cbl is compensating for the diminished SOD activity but also that it is capable of doing so in the mitochondria.
6.2.3 Cbl as general antioxidant.
•- Our work focused on the protective effects of Cbl against O2 -induced cell
damage. The potential benefits of Cbl supplementation on oxidative stress induced by
other ROS is also of importance. Prior to investigating the effect of Cbl supplementation
in a biological system challenged with a specific oxidative insult, the chemistry of the
direct reactions of Cbl species with these ROS should be studied where necessary. A
good understanding of the rates and mechanisms of these reactions would provide the
necessary basis to assess the potential biological importance of Cbl scavenging of these
species.
In order to determine if Cbl is indeed a broad spectrum scavenger, the ability of
Cbl to reduce intracellular levels of other ROS and to prevent ROS-induced cellular
dysfunction could be studied. Based on the hypothesis that oxidative stress is the unifying
feature of CVD, antioxidants should be effective for the prevention and treatment of
CVD. In the field of vascular biology, several epidemiologic studies support this
hypothesis (296). Therefore, a logical step in order to test the antioxidant and vasculo-
protective effects of Cbls is to evaluate the benefits of Cbl supplementation in an animal
142
model of cardiovascular disease. Atherosclerosis is one of the most well studied vasculopathy for which there is strong experimental evidence implicating oxidative stress in the pathogenesis of the disease. Apolipoprotein E (apoE) knockout mice spontaneously develop atherosclerotic lesions (297), and this model has been used used to study the effect of diet on the development and progression of atherosclerosis. This would, therefore, be an ideal model to study the benefits of Cbl supplementation on the prevention of oxidative stress-related vasculopathies.
143
REFERENCES
1. Raju, T.N., The Nobel chronicles. 1934: George Hoyt Whipple (1878-1976); George Richard Minot (1885-1950); William Perry Murphy (1892-1987). Lancet, 1999. 353(9148): p. 247. 2. Pruthi, R.K. and A. Tefferi, Pernicious anemia revisited. Mayo Clin Proc, 1994. 69(2): p. 144-50. 3. Whipple, G.H. and F.S. Robscheit-Robbins, Favorable influence of liver, heart, and skeletal muscle in diet on blood regeneration in anemia. Am J Physiol, 1925. 72(408- 418). 4. Minot, G.R. and W.P. Murphy, Treatment of pernicious anemia by a special diet. . J Am Med Assoc, 1926. 87: p. 470-476. 5. Rickes, E.L., N.G. Brink, F.R. Koniuszy, T.R. Wood, and K. Folkers, Comparative Data on Vitamin B12 From Liver and From a New Source, Streptomyces griseus. Science, 1948. 108(2814): p. 634-635. 6. Rickes, E.L., N.G. Brink, F.R. Koniuszy, T.R. Wood, and K. Folkers, Vitamin B12, A Cobalt Complex. Science, 1948. 108(2797): p. 134. 7. Rickes, E.L., N.G. Brink, F.R. Koniuszy, T.R. Wood, and K. Folkers, Crystalline Vitamin B12. Science, 1948. 107(2781): p. 396-397. 8. Smith, E.L., Presence of cobalt in the anti-pernicious anaemia factor. Nature, 1948. 162(4108): p. 144. 9. Smith, E.L., Purification of anti-pernicious anaemia factors from liver. Nature, 1948. 161(4095): p. 638. 10. Hodgkin, D.C., J. Kamper, M. Mackay, J. Pickworth, K.N. Trueblood, and J.G. White, Structure of vitamin B12. Nature, 1956. 178(4524): p. 64-6. 11. Shampo, M.A. and R.A. Kyle, Dorothy Hodgkin--1964 Nobel laureate for work on vitamin B12. Mayo Clin Proc, 2002. 77(5): p. 403. 12. Chemistry, I.U.o.P.a.A. and I.U.o. Biochemistry, Nomenclature of Corrinoids. Pure & Appl Chem, 1976. 48(4): p. 495-502. 13. Brown, K.L., Chemistry and Enzymology of Vitamin B12. Chem Rev, 2005. 105(6): p. 2075-2149. 14. Banerjee, R., Radical carbon skeleton rearrangements: catalysis by coenzyme B12- dependent mutases. Chem Rev, 2003. 103(6): p. 2083-94. 15. Rossi, M., J.P. Glusker, L. Randaccio, M.F. Summers, P.J. Toscano, and L.G. Marzilli, The structure of a B12 coenzyme: methylcobalamin studies by x-ray and NMR methods. J Am Chem Soc, 1985. 107(6): p. 1729-1738. 16. Krautler, B., Vitamin B12: chemistry and biochemistry. Biochem Soc Trans, 2005. 33(Pt 4): p. 806-10. 17. Barker, H.A., H. Weissbach, and R.D. Smyth, A Coenzyme Containing Pseudovitamin B(12). Proc Natl Acad Sci U S A, 1958. 44(11): p. 1093-7. 18. Lenhert, P.G. and D.C. Hodgkin, Structure of the 5,6-dimethyl-benzimidazolylcobamide coenzyme. Nature, 1961. 192: p. 937-8. 19. Guest, J.R., S. Friedman, D.D. Woods, and E.L. Smith, A methyl analogue of cobamide coenzyme in relation to methionine synthesis by bacteria. Nature, 1962. 195: p. 340-2. 143 144
20. Banerjee, R. and S.W. Ragsdale, The many faces of vitamin B12: catalysis by cobalamin- dependent enzymes. Annu Rev Biochem, 2003. 72: p. 209-47. 21. Banerjee, R., The Yin-Yang of cobalamin biochemistry. Chem Biol, 1997. 4(3): p. 175-86. 22. Gulati, S., Z. Chen, L.C. Brody, D.S. Rosenblatt, and R. Banerjee, Defects in auxiliary redox proteins lead to functional methionine synthase deficiency. J Biol Chem, 1997. 272(31): p. 19171-5. 23. Brada, N., M.M. Gordon, J. Wen, and D.H. Alpers, Transfer of cobalamin from intrinsic factor to transcobalamin II. J Nutr Biochem, 2001. 12(4): p. 200-206. 24. Nexo, E., Cobalamin Binding Proteins, in Vitamin B12 and B12-proteins : lectures presented at the 4th European Symposium on Vitamin B12 and B12-Proteins, B. Krautler, D. Arigoni, and B. Golding, Editors. 1998, Wiley-VCH: Weinheim. p. 461-76. 25. Cannon, M.J., D.G. Myszka, J.D. Bagnato, D.H. Alpers, F.G. West, and C.B. Grissom, Equilibrium and kinetic analyses of the interactions between vitamin B(12) binding proteins and cobalamins by surface plasmon resonance. Anal Biochem, 2002. 305(1): p. 1-9. 26. Quadros, E.V., Y. Nakayama, and J.M. Sequeira, The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin. Blood, 2009. 113(1): p. 186-92. 27. Rosenblatt, D.S. and W.A. Fenton, Inherited Disorders of Folate and Cobalamin Transport and Metabolism, in The Metabolic & Molecular Bases of Inherited Disease, S.R. Scriver, et al., Editors. 2001, McGraw Hill: New York. p. 3897-3933. 28. Rossenblatt, D., Inborn Errors of Folate and Cobalamin Metabolism, in Homocysteine in Health and Disease, R. Carmel and D. Jacobsen, Editors. 2001, University Press: Cambridge. p. 244-58. 29. Rosenblatt, D.S., A. Hosack, N.V. Matiaszuk, B.A. Cooper, and R. Laframboise, Defect in vitamin B12 release from lysosomes: newly described inborn error of vitamin B12 metabolism. Science, 1985. 228(4705): p. 1319-21. 30. Kim, J., L. Hannibal, C. Gherasim, D.W. Jacobsen, and R. Banerjee, A human B12 trafficking protein uses glutathione transferase activity for processing alkylcobalamins. J Biol Chem, 2009. 31. Coelho, D., T. Cuormala, M. Stucki, J.P. Lerner-Ellis, D.S. Rosenblatt, R.F. Newbold, M.R. Baumgartner, and B. Fowler, Gene identification for the cblD Defect of Vitamin B12 Metabolism. N Engl J Med, 2008. 358(14): p. 1454-1464. 32. Hannibal, L., J. Kim, N.E. Brasch, S. Wang, D.S. Rosenblatt, R. Banerjee, and D.W. Jacobsen, Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product. Mol Genet Metab, 2009. 97(4): p. 260-6. 33. Kim, J., C. Gherasim, and R. Banerjee, Decyanation of vitamin B12 by a trafficking chaperone. Proc Natl Acad Sci U S A, 2008. 105(38): p. 14551-4. 34. Lerner-Ellis, J.P., J.C. Tirone, P.D. Pawelek, C. Dore, J.L. Atkinson, D. Watkins, C.F. Morel, T.M. Fujiwara, E. Moras, A.R. Hosack, G.V. Dunbar, H. Antonicka, V. Forgetta, C.M. Dobson, D. Leclerc, R.A. Gravel, E.A. Shoubridge, J.W. Coulton, P. Lepage, J.M. Rommens, K. Morgan, and D.S. Rosenblatt, Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type. Nat Genet, 2006. 38(1): p. 93- 100. 35. Yamanishi, M., T. Labunska, and R. Banerjee, Mirror "base-off" conformation of coenzyme B12 in human adenosyltransferase and its downstream target, methylmalonyl- CoA mutase. J Am Chem Soc, 2005. 127(2): p. 526-7.
145
36. Yamanishi, M., M. Vlasie, and R. Banerjee, Adenosyltransferase: an enzyme and an escort for coenzyme B12? Trends Biochem Sci, 2005. 30(6): p. 304-8. 37. Leal, N.A., S.D. Park, P.E. Kima, and T.A. Bobik, Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant. J Biol Chem, 2003. 278(11): p. 9227-34. 38. Dobson, C.M., T. Wai, D. Leclerc, H. Kadir, M. Narang, J.P. Lerner-Ellis, T.J. Hudson, D.S. Rosenblatt, and R.A. Gravel, Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria. Hum Mol Genet, 2002. 11(26): p. 3361-9. 39. Gulati, S., P. Baker, Y.N. Li, B. Fowler, W. Kruger, L.C. Brody, and R. Banerjee, Defects in human methionine synthase in cblG patients. Hum Mol Genet, 1996. 5(12): p. 1859-65. 40. Jacobsen, D.W., E.H. Pezacka, and K.L. Brown, The inhibition of corrinoid-catalyzed oxidation of mercaptoethanol by methyl iodide: mechanistic implications. J Inorg Biochem, 1993. 50(1): p. 47-63. 41. Jacobsen, D.W., L.S. Troxell, and K.L. Brown, Catalysis of Thiol oxidation by Cobalamins and Cobinamides: Reaction Products and Kinetics. Biochem, 1984. 23: p. 2017-25. 42. McCaddon, A., A method for treating or preventing a functional vitamin B12 deficiency in an individual and medical compositions for use in said method. 2002, (Cobalz Limited, UK). Wales. p. 59 pp. 43. Sies, H., Glutathione and its role in cellular functions. Free Radic Biol Med, 1999. 27(9- 10): p. 916-21. 44. Xia, L., A.G. Cregan, L.A. Berben, and N.E. Brasch, Studies on the formation of glutathionylcobalamin: any free intracellular aquacobalamin is likely to be rapidly and irreversibly converted to glutathionylcobalamin. Inorg Chem, 2004. 43(21): p. 6848-57. 45. Pezacka, E.H., Identification and characterization of two enzymes involved in the intracellular metabolism of cobalamin. Cyanocobalamin beta-ligand transferase and microsomal cob(III)alamin reductase. Biochim Biophys Acta, 1993. 1157(2): p. 167-77. 46. Pezacka, E.H., D.W. Jacobsen, K. Luce, and R. Green, Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation. Biochem Biophys Res Commun, 1992. 184(2): p. 832-9. 47. Pezacka, E., R. Green, and D.W. Jacobsen, Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes. Biochem Biophys Res Commun, 1990. 169(2): p. 443-50. 48. Jacobsen, D.W. and R. Green, Glutathionylcobalamin (GS-Cbl) is found in normal and neoplasic cells. Blood, 1986. 68(Suppl. 1): p. 247a. 49. Jacobsen, D., C. Lee-Deninson, K. Kuce, and R. Green, Glutathionylcobalamin (GS-Cbl) is found in cultured and ascites leukemia L1210 cells. Fed. Proc., 1987. 46: p. 1005. 50. Hannibal, L., A. Axhemi, A.V. Glushchenko, E. Suarez-Moreira, N.E. Brasch, and D.W. Jacobsen, Accurate assessment and identification of naturally occurring cellular cobalamins. Clin Chem Lab Med, 2008. 51. McKeever, M.P., D.G. Weir, A. Molloy, and J.M. Scott, Betaine-homocysteine methyltransferase: organ distribution in man, pig and rat and subcellular distribution in the rat. Clin Sci (Lond), 1991. 81(4): p. 551-6.
146
52. Molloy, A.M. and D.G. Weir, Homocysteine and the Nervous System, in Homocysteine in Health and Disease, D.W. Jacobsen and R. Carmel, Editors. 2001, Cambridge University Press: Cambridge. 53. McCaddon, A., G. Davies, P. Hudson, S. Tandy, and H. Cattell, Total serum homocysteine in senile dementia of Alzheimer type. Int J Geriatr Psychiatry, 1998. 13(4): p. 235-9. 54. Wolters, M., A. Strohle, and A. Hahn, Cobalamin: a critical vitamin in the elderly. Prev Med, 2004. 39(6): p. 1256-66. 55. Allen, L.H., Causes of vitamin B12 and folate deficiency. Food Nutr Bull, 2008. 29(2 Suppl): p. S20-34; discussion S35-7. 56. Hvas, A.M. and E. Nexo, Diagnosis and treatment of vitamin B12 deficiency--an update. Haematologica, 2006. 91(11): p. 1506-12. 57. Solomon, L.R., Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing. Blood, 2005. 105(3): p. 978-85. 58. Refsum, H., C. Johnston, A.B. Guttormsen, and E. Nexo, Holotranscobalamin and total transcobalamin in human plasma: determination, determinants, and reference values in healthy adults. Clin Chem, 2006. 52(1): p. 129-37. 59. Droge, W., Free radicals in the physiological control of cell function. Physiol Rev, 2002. 82(1): p. 47-95. 60. McCord, J.M. and B.A. Omar, Sources of free radicals. Toxicol Ind Health, 1993. 9(1-2): p. 23-37. 61. McCord, J.M. and M.A. Edeas, SOD, oxidative stress and human pathologies: a brief history and a future vision. Biomed Pharmacother, 2005. 59(4): p. 139-42. 62. Muscoli, C., S. Cuzzocrea, D.P. Riley, J.L. Zweier, C. Thiemermann, Z.Q. Wang, and D. Salvemini, On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies. Br J Pharmacol, 2003. 140(3): p. 445-60. 63. Buetler, T.M., A. Krauskopf, and U.T. Ruegg, Role of superoxide as a signaling molecule. News Physiol Sci, 2004. 19: p. 120-3. 64. Fridovich, I., Superoxide dismutases. An adaptation to a paramagnetic gas. J Biol Chem, 1989. 264(14): p. 7761-4. 65. Schroder, E. and P. Eaton, Hydrogen peroxide as an endogenous mediator and exogenous tool in cardiovascular research: issues and considerations. Curr Opin Pharmacol, 2008. 8(2): p. 153-9. 66. Saitoh, S., T. Kiyooka, P. Rocic, P.A. Rogers, C. Zhang, A. Swafford, G.M. Dick, C. Viswanathan, Y. Park, and W.M. Chilian, Redox-dependent coronary metabolic dilation. Am J Physiol Heart Circ Physiol, 2007. 293(6): p. H3720-5. 67. Kim, Y.M., K.E. Kim, G.Y. Koh, Y.S. Ho, and K.J. Lee, Hydrogen peroxide produced by angiopoietin-1 mediates angiogenesis. Cancer Res, 2006. 66(12): p. 6167-74. 68. Ushio-Fukai, M. and R.W. Alexander, Reactive oxygen species as mediators of angiogenesis signaling: role of NAD(P)H oxidase. Mol Cell Biochem, 2004. 264(1-2): p. 85-97. 69. Roy, S., S. Khanna, and C.K. Sen, Redox regulation of the VEGF signaling path and tissue vascularization: Hydrogen peroxide, the common link between physical exercise and cutaneous wound healing. Free Radic Biol Med, 2008. 44(2): p. 180-92. 70. Groeger, G., C. Quiney, and T.G. Cotter, Hydrogen peroxide as a cell-survival signaling molecule. Antioxid Redox Signal, 2009. 11(11): p. 2655-71.
147
71. Gutteridge, J.M. and B. Halliwell, Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci, 2000. 899: p. 136-47. 72. Nitric Oxide Biology and Pathobiology, L.J. Ignarro, Editor. 2000, Academic Press: New York. 73. Peluffo, G. and R. Radi, Biochemistry of protein tyrosine nitration in cardiovascular pathology. Cardiovasc Res, 2007. 75(2): p. 291-302. 74. Weil, M., R. Abeles, A. Nachmany, V. Gold, and E. Michael, Folic acid rescues nitric oxide-induced neural tube closure defects. Cell Death Differ, 2004. 11(3): p. 361-3. 75. Brouwer, M., W. Chamulitrat, G. Ferruzzi, D.L. Sauls, and J.B. Weinberg, Nitric oxide interactions with cobalamins: biochemical and functional consequences. Blood, 1996. 88(5): p. 1857-64. 76. Jiang, F., C.G. Li, and M.J. Rand, Effect of hydroxocobalamin on vasodilatations to nitrergic transmitter, nitric oxide and endothelium-derived relaxing factor in guinea-pig basilar artery. Eur J Pharmacol, 1997. 340(2-3): p. 181-6. 77. Greenberg, S.S., J. Xie, J.M. Zatarain, D.R. Kapusta, and M.J. Miller, Hydroxocobalamin (vitamin B12a) prevents and reverses endotoxin-induced hypotension and mortality in rodents: role of nitric oxide. J Pharmacol Exp Ther, 1995. 273(1): p. 257-65. 78. Jenkinson, K.M., J.J. Reid, and M.J. Rand, Hydroxocobalamin and haemoglobin differentiate between exogenous and neuronal nitric oxide in the rat gastric fundus. Eur J Pharmacol, 1995. 275(2): p. 145-52. 79. Rajanayagam, M.A.S., S.G. Li, and M.J. Rand, Differential effects of hydroxocobalamin on nitric oxide-mediated relaxations in rat aorta and anococcygeus. Br J Pharmacol, 1993. 108(1): p. 2-5. 80. Rochelle, L.G., S.J. Morana, H. Kruszyna, M.A. Russell, D.E. Wilcox, and R.P. Smith, Interactions between hydroxocobalamin and nitric oxide (NO): evidence for a redox reaction between NO and reduced cobalamin and reversible NO binding to oxidized cobalamin. J Pharmacol Exp Ther, 1995. 275(1): p. 48-52. 81. Hannibal, L., C. Smith, D.W. Jacobsen, and N.E. Brasch, A Simple and Efficient Method to Synthesize Nitrosylcobalamin from Aquacobalamin and 2-(N,N-Diethylamino)- diazenolate 2-oxide. J Am Chem Soc, 2006. submitted. 82. Selcuki, C., R. van Eldik, and T. Clark, NO binding to cobalamin: influence of the metal oxidation state. Inorg Chem, 2004. 43(9): p. 2828-33. 83. Wolak, M., G. Stochel, M. Hamza, and R. van Eldik, Aquacobalamin (vitamin B12a) does not bind NO in aqueous solution. Nitrite impurities account for observed reaction. Inorg Chem, 2000. 39(10): p. 2018-9. 84. Zheng, D. and R.L. Birke, Spectroscopic evidence for nitric oxide binding with cob(II)alamin. J Am Chem Soc, 2001. 123(19): p. 4637-8. 85. Suarez-Moreira, E., L. Hannibal, C.A. Smith, R.A. Chavez, D.W. Jacobsen, and N.E. Brasch, A simple, convenient method to synthesize cobalamins: synthesis of homocysteinylcobalamin, N-acetylcysteinylcobalamin, 2-N-acetylamino-2- carbomethoxyethanethiolatocobalamin, sulfitocobalamin and nitrocobalamin. Dalton Trans, 2006(44): p. 5269-77. 86. Knapton, L. and H.M. Marques, Probing the nature of the Co(III) ion in cobalamins: a comparison of the reaction of aquacobalamin (vitamin B12a) and aqua-10- chlorocobalamin with some anionic and N-donor ligands. Dalton Trans, 2005(5): p. 889- 95. 87. Marques, H.M. and L. Knapton, J Chem Soc, Dalton Trans, 1997. 20: p. 3827-3833.
148
88. Wolak, M., A. Zahl, T. Schneppensieper, G. Stochel, and R. van Eldik, Kinetics and mechanism of the reversible binding of nitric oxide to reduced cobalamin B(12r) (Cob(II)alamin). J Am Chem Soc, 2001. 123(40): p. 9780-91. 89. Birch, C.S., N.E. Brasch, A. McCaddon, and J.H. Williams, A novel role for vitamin B(12): Cobalamins are intracellular antioxidants in vitro. Free Radic Biol Med, 2009. 47(2): p. 184-8. 90. Higashi-Okai, K., H. Nagino, K. Yamada, and Y. Okai, Antioxidant and prooxidant activities of B group vitamins in lipid peroxidation. J Uoeh, 2006. 28(4): p. 359-68. 91. Mukherjee, S., D. Das, M. Mukherjee, A.S. Das, and C. Mitra, Synergistic effect of folic acid and vitamin B12 in ameliorating arsenic-induced oxidative damage in pancreatic tissue of rat. J Nutr Biochem, 2006. 17(5): p. 319-27. 92. Faxon, D.P., V. Fuster, P. Libby, J.A. Beckman, W.R. Hiatt, R.W. Thompson, J.N. Topper, B.H. Annex, J.H. Rundback, R.P. Fabunmi, R.M. Robertson, and J. Loscalzo, Atherosclerotic Vascular Disease Conference: Writing Group III: pathophysiology. Circulation, 2004. 109(21): p. 2617-25. 93. Rollins, B.J., Chemokines. Blood, 1997. 90(3): p. 909-28. 94. Rollins, B.J., Chemokines and atherosclerosis: what Adam Smith has to say about vascular disease. J Clin Invest, 2001. 108(9): p. 1269-71. 95. Creager, M.A., J.P. Cooke, M.E. Mendelsohn, S.J. Gallagher, S.M. Coleman, J. Loscalzo, and V.J. Dzau, Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest, 1990. 86(1): p. 228-34. 96. Lieberman, E.H., M.D. Gerhard, A. Uehata, A.P. Selwyn, P. Ganz, A.C. Yeung, and M.A. Creager, Flow-induced vasodilation of the human brachial artery is impaired in patients <40 years of age with coronary artery disease. Am J Cardiol, 1996. 78(11): p. 1210-4. 97. Forstermann, U. and T. Munzel, Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 2006. 113(13): p. 1708-14. 98. Madamanchi, N.R., A. Vendrov, and M.S. Runge, Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol, 2005. 25(1): p. 29-38. 99. Mohazzab, K.M., P.M. Kaminski, and M.S. Wolin, NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol, 1994. 266(6 Pt 2): p. H2568-72. 100. Azumi, H., N. Inoue, Y. Ohashi, M. Terashima, T. Mori, H. Fujita, K. Awano, K. Kobayashi, K. Maeda, K. Hata, T. Shinke, S. Kobayashi, K. Hirata, S. Kawashima, H. Itabe, Y. Hayashi, S. Imajoh-Ohmi, H. Itoh, and M. Yokoyama, Superoxide generation in directional coronary atherectomy specimens of patients with angina pectoris: important role of NAD(P)H oxidase. Arterioscler Thromb Vasc Biol, 2002. 22(11): p. 1838-44. 101. Maack, C., T. Kartes, H. Kilter, H.J. Schafers, G. Nickenig, M. Bohm, and U. Laufs, Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment. Circulation, 2003. 108(13): p. 1567-74. 102. De Biase, L., P. Pignatelli, L. Lenti, G. Tocci, F. Piccioni, S. Riondino, F.M. Pulcinelli, S. Rubattu, M. Volpe, and F. Violi, Enhanced TNF alpha and oxidative stress in patients with heart failure: effect of TNF alpha on platelet O2- production. Thromb Haemost, 2003. 90(2): p. 317-25. 103. Heymes, C., J.K. Bendall, P. Ratajczak, A.C. Cave, J.L. Samuel, G. Hasenfuss, and A.M. Shah, Increased myocardial NADPH oxidase activity in human heart failure. J Am Coll Cardiol, 2003. 41(12): p. 2164-71.
149
104. Vaziri, N.D., Causal link between oxidative stress, inflammation, and hypertension. Iran J Kidney Dis, 2008. 2(1): p. 1-10. 105. El Kossi, M.M. and M.M. Zakhary, Oxidative stress in the context of acute cerebrovascular stroke. Stroke, 2000. 31(8): p. 1889-92. 106. Alexandrova, M.L., P.G. Bochev, V.I. Markova, B.G. Bechev, M.A. Popova, M.P. Danovska, and V.K. Simeonova, Changes in phagocyte activity in patients with ischaemic stroke. Luminescence, 2001. 16(6): p. 357-65. 107. Klee, G.G., Cobalamin and folate evaluation: measurement of methylmalonic acid and homocysteine vs vitamin B(12) and folate. Clin Chem, 2000. 46(8 Pt 2): p. 1277-83. 108. Jacobsen, D.W., Homocysteine and vitamins in cardiovascular disease. Clin Chem, 1998. 44(8 Pt 2): p. 1833-43. 109. Brown, C.A., K.Q. McKinney, J.S. Kaufman, R.A. Gravel, and R. Rozen, A common polymorphism in methionine synthase reductase increases risk of premature coronary artery disease. J Cardiovasc Risk, 2000. 7(3): p. 197-200. 110. Selhub, J., P.F. Jacques, I.H. Rosenberg, G. Rogers, B.A. Bowman, E.W. Gunter, J.D. Wright, and C.L. Johnson, Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991-1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med, 1999. 131(5): p. 331-9. 111. Jacobsen, D., Cellular Mechanisms of Homocysteine Pathgenesis in Atherosclerosis, in Homocysteine in Health and Disease, R. Carmel and D. Jacobsen, Editors. 2001, University Press: Cambridge. p. 425-40. 112. Chen, C., M.E. Halkos, S.M. Surowiec, B.S. Conklin, P.H. Lin, and A.B. Lumsden, Effects of homocysteine on smooth muscle cell proliferation in both cell culture and artery perfusion culture models. J Surg Res, 2000. 88(1): p. 26-33. 113. Dennis, V.W. and K. Robinson, Homocysteinemia and vascular disease in end-stage renal disease. Kidney Int Suppl, 1996. 57: p. S11-7. 114. Carmel, R., R. Green, D.S. Rosenblatt, and D. Watkins, Update on cobalamin, folate, and homocysteine. Hematology (Am Soc Hematol Educ Program), 2003: p. 62-81. 115. Taylor, B.V., G.Y. Oudit, and M. Evans, Homocysteine, vitamins, and coronary artery disease. Comprehensive review of the literature. Can Fam Physician, 2000. 46: p. 2236- 45. 116. Verhoef, P., M.J. Stampfer, J.E. Buring, J.M. Gaziano, R.H. Allen, S.P. Stabler, R.D. Reynolds, F.J. Kok, C.H. Hennekens, and W.C. Willett, Homocysteine metabolism and risk of myocardial infarction: relation with vitamins B6, B12, and folate. Am J Epidemiol, 1996. 143(9): p. 845-59. 117. Liaugaudas, G., P.F. Jacques, J. Selhub, I.H. Rosenberg, and A.G. Bostom, Renal insufficiency, vitamin B(12) status, and population attributable risk for mild hyperhomocysteinemia among coronary artery disease patients in the era of folic acid- fortified cereal grain flour. Arterioscler Thromb Vasc Biol, 2001. 21(5): p. 849-51. 118. Garrow, T.A., Purification, kinetic properties, and cDNA cloning of mammalian betaine- homocysteine methyltransferase. J Biol Chem, 1996. 271(37): p. 22831-8. 119. Finkelstein, J.D., Methionine metabolism in mammals. J Nutr Biochem, 1990. 1(5): p. 228-37. 120. Chen, P., R. Poddar, E.V. Tipa, P.M. Dibello, C.D. Moravec, K. Robinson, R. Green, W.D. Kruger, T.A. Garrow, and D.W. Jacobsen, Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease. Adv Enzyme Regul, 1999. 39: p. 93-109.
150
121. Wang, J., N.P. Dudman, D.E. Wilcken, and J.F. Lynch, Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease. Atherosclerosis, 1992. 97(1): p. 97-106. 122. Jacobsen, D.W., Biochemistry and Metabolism of Homocysteine, in Homocysteine and Vascular Disease, K. Robinson, Editor. 2000, Kluwer Academic Publishers: Dordrecht. p. 15-39. 123. Quadros, E.V., S.P. Rothenberg, and E.A. Jaffe, Endothelial cells from human umbilical vein secrete functional transcobalamin II. Am J Physiol, 1989. 256(2 Pt 1): p. C296-303. 124. Nexo, E., A.M. Hvas, O. Bleie, H. Refsum, S.N. Fedosov, S.E. Vollset, J. Schneede, J.E. Nordrehaug, P.M. Ueland, and O.K. Nygard, Holo-transcobalamin is an early marker of changes in cobalamin homeostasis. A randomized placebo-controlled study. Clin Chem, 2002. 48(10): p. 1768-71. 125. Solomon, L.R., Disorders of cobalamin (vitamin B12) metabolism: emerging concepts in pathophysiology, diagnosis and treatment. Blood Rev, 2007. 21(3): p. 113-30. 126. Oltean, S. and R. Banerjee, A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase. J Biol Chem, 2005. 280(38): p. 32662-8. 127. Watanabe, T., R. Kaji, N. Oka, W. Bara, and J. Kimura, Ultra-high dose methylcobalamin promotes nerve regeneration in experimental acrylamide neuropathy. J Neurol Sci, 1994. 122(2): p. 140-3. 128. Caram-Salas, N.L., G. Reyes-Garcia, R. Medina-Santillan, and V. Granados-Soto, Thiamine and cyanocobalamin relieve neuropathic pain in rats: synergy with dexamethasone. Pharmacology, 2006. 77(2): p. 53-62. 129. Kaji, R., M. Kodama, A. Imamura, T. Hashida, N. Kohara, M. Ishizu, K. Inui, and J. Kimura, Effect of ultrahigh-dose methylcobalamin on compound muscle action potentials in amyotrophic lateral sclerosis: a double-blind controlled study. Muscle Nerve, 1998. 21(12): p. 1775-8. 130. Nourhashemi, F., S. Gillette-Guyonnet, S. Andrieu, A. Ghisolfi, P.J. Ousset, H. Grandjean, A. Grand, J. Pous, B. Vellas, and J.L. Albarede, Alzheimer disease: protective factors. Am J Clin Nutr, 2000. 71(2): p. 643S-649S. 131. McGeer, P.L. and E.G. McGeer, Inflammation, autotoxicity and Alzheimer disease. Neurobiol Aging, 2001. 22(6): p. 799-809. 132. Miller, J.W., Vitamin B12 deficiency, tumor necrosis factor-alpha, and epidermal growth factor: a novel function for vitamin B12? Nutr Rev, 2002. 60(5 Pt 1): p. 142-4. 133. Yamashiki, M., A. Nishimura, and Y. Kosaka, Effects of methylcobalamin (vitamin B12) on in vitro cytokine production of peripheral blood mononuclear cells. J Clin Lab Immunol, 1992. 37(4): p. 173-82. 134. Flynn, M.A., W. Irvin, and G. Krause, The effect of folate and cobalamin on osteoarthritic hands. J Am Coll Nutr, 1994. 13(4): p. 351-6. 135. Volkov, I., I. Rudoy, and Y. Press, Successful treatment of chronic erythema nodosum with vitamin B12. J Am Board Fam Pract, 2005. 18(6): p. 567-9. 136. Peracchi, M., F. Bamonti Catena, M. Pomati, M. De Franceschi, and G. Scalabrino, Human cobalamin deficiency: alterations in serum tumour necrosis factor-alpha and epidermal growth factor. Eur J Haematol, 2001. 67(2): p. 123-7. 137. Wheatley, C., A scarlet pimpernel for the resolution of inflammation? The role of supra- therapeutic doses of cobalamin, in the treatment of systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic or traumatic shock. Med Hypotheses, 2006. 67(1): p. 124-42.
151
138. Wheatley, C., The return of the Scarlet Pimpernel: cobalamin in inflammation II - cobalamins can both selectively promote all three nitric oxide synthases (NOS), particularly iNOS and eNOS, and, as needed, selectively inhibit iNOS and nNOS. J Nutr Environ Med, 2007. 16(3-4): p. 181-211. 139. Wheatley, C., Cobalamin in inflammation III - glutathionylcobalamin and methylcobalamin/adenosylcobalamin coenzymes: the sword in the stone? How cobalamin may directly regulate the nitric oxide synthases. J Nutr Environ Med, 2007. 16(3-4): p. 212-226. 140. Cregan, A.G., N.E. Brasch, and R. van Eldik, Thermodynamic and kinetic studies on the reaction between the vitamin B12 derivative beta-(N-methylimidazolyl)cobalamin and N- methylimidazole: ligand displacement at the alpha axial site of cobalamins. Inorg Chem, 2001. 40(7): p. 1430-8. 141. Linnell, J.C., A.V. Hoffbrand, H.A. Hussein, I.J. Wise, and D.M. Matthews, Tissue distribution of coenzyme and other forms of vitamin B12 in control subjects and patients with pernicious anaemia. Clin Sci Mol Med, 1974. 46(2): p. 163-72. 142. Gimsing, P. and E. Nexo, The forms of cobalamin in biological material, in The Cobalamins, C.A. Hall, Editor. 1983, Churchill Livingstone: New York. 143. Gimsing, P., E. Nexo, and E. Hippe, Determination of cobalamins in biological material. II. The cobalamins in human plasma and erythrocytes after desalting on nonpolar adsorbent material, and separation by one-dimensional thin-layer chromatography. Anal Biochem, 1983. 129(2): p. 296-304. 144. Jacobsen, D.W., R. Green, E.V. Quadros, and Y.D. Montejano, Rapid analysis of cobalamin coenzymes and related corrinoid analogs by high-performance liquid chromatography. Anal Biochem, 1982. 120(2): p. 394-403. 145. Anes, J.M., R.A. Beck, J.J. Brink, and R.J. Goldberg, Nitritocobalamin and nitrosocobalamin may be confused with sulfitocobalamin using cation-exchange chromatography. J Chromatogr B Biomed Appl, 1994. 660(1): p. 180-5. 146. Beck, R.A., J.M. Anes, L.M. Savini, and R.A. Mateer, Picomolar quantitation of free sulfite in foods by means of [57Co]hydroxocobalamin and radiometric chromatography of [57Co]sulfitocobalamin. Method, applications and significance of coexisting sulfides. J Chromatogr A, 2000. 881(1-2): p. 345-56. 147. Farquharson, J. and J.F. Adams, Conversion of hydroxo(aquo) cobalamin to sulfitocobalamin in the absence of light: a reaction of importance in the identification of the forms of vitamin B12, with possible clinical significance. Am J Clin Nutr, 1977. 30(10): p. 1617-22. 148. Farquharson, J. and J.F. Adams, The forms of vitamin B12 in foods. Br J Nutr, 1976. 36(1): p. 127-36. 149. Begley, J.A. and C.A. Hall, Overestimation of cyanocobalamin due to coelution of sulfitocobalamin on SP-Sephadex C-25. J Chromatogr, 1979. 177(2): p. 360-2. 150. Takenaka, S., K. Takubo, F. Watanabe, T. Tanno, S. Tsuyama, Y. Nanano, and Y. Tamura, Occurrence of coenzyme forms of vitamin B12 in a cultured purple laver (Porphyla yezoensis). Biosci Biotechnol Biochem, 2003. 67(11): p. 2480-2. 151. Takenaka, S., S. Sugiyama, S. Ebara, E. Miyamoto, K. Abe, Y. Tamura, F. Watanabe, S. Tsuyama, and Y. Nakano, Feeding dried purple laver (nori) to vitamin B12-deficient rats significantly improves vitamin B12 status. Br J Nutr, 2001. 85(6): p. 699-703. 152. Smith, E.L., K.H. Fantes, S. Ball, J.G. Waller, W.B. Emery, W.K. Anslow, and A.D. Walker, B12 vitamins (cobalamins). I. Vitamins B12c and B12d. Biochem J, 1952. 52(3): p. 389-95.
152
153. Koyama, K., A. Yoshida, A. Takeda, K. Morozumi, T. Fujinami, and N. Tanaka, Abnormal cyanide metabolism in uremic patients. Nephrol Dial Transplant, 1997. 12(8): p. 1622-1628. 154. Wagner, F. and K. Bernhauer, New aspects of the structure of corrinoid coenzymes. Ann N Y Acad Sci, 1964. 112(2): p. 580-9. 155. Zheng, D. and R.L. Birke, The Reaction of Nitric Oxide with Glutathionylcobalamin. J Am Chem Soc, 2002. 124(31): p. 9066-9067. 156. Watson, W.P., T. Munter, and B.T. Golding, A New Role for Glutathione: Protection of Vitamin B12 from Depletion by Xenobiotics. Chem Res Toxicol, 2004. 17(12): p. 1562- 1567. 157. Brasch, N.E., T.L. Hsu, K.M. Doll, and R.G. Finke, Synthesis and characterization of isolable thiolatocobalamin complexes relevant to coenzyme B12-dependent ribonucleoside triphosphate reductase. J Inorg Biochem, 1999. 76(3-4): p. 197-209. 158. Hsu, T.-L.C., N.E. Brasch, and R.G. Finke, The Second Isolable B12-Thiolate Complex, (Pentafluorophenylthiolato)cobalamin: Synthesis and Characterization. Inorg Chem, 1998. 37(20): p. 5109-5116. 159. Brasch, N.E. and L. Xia, Method of synthesis of b-thiolato cobalamin nucleoside compounds. 2004: USA. p. 5 pp. 160. Brown, K.L., X. Zou, S.R. Savon, and D.W. Jacobsen, Heteronuclear nuclear magnetic resonance studies of cobalt corrinoids. 15. The structure of glutathionylcobalamin: A proton and carbon-13 two-dimensional nuclear magnetic resonance study at 600 MHz. Biochemistry, 1993. 32(33): p. 8421-8. 161. Scheuring, E.M., I. Sagi, and M.R. Chance, Sulfur-containing cobalamins: X-ray absorption spectroscopic characterization. Biochemistry, 1994. 33(20): p. 6310-5. 162. Ngandu, L., D. Robin, A. El Kasmi, and D. Lexa, Vitamin B12-transport protein interaction: electrochemistry of aquo- and glutathionyl-cobalamins adsorbed on carbon electrodes; role of the nucleotide chain. Inorg Chim Acta, 1999. 292(2): p. 204-212. 163. McCaddon, A., B. Regland, P. Hudson, and G. Davies, Functional vitamin B(12) deficiency and Alzheimer disease. Neurology, 2002. 58(9): p. 1395-9. 164. Dubnoff, J.W., A cobalamin glutathione complex. Biochem Biophys Res Commun, 1964. 16(5): p. 484-8. 165. Dolphin, D. and A.W. Johnson, The Properties of Cobalamins with Sulphur-Containing Ligands. J Chem Soc, 1965. 65: p. 2174-81. 166. Dolphin, D.H. and A.W. Johnson, The Reaction of Cobalamins with Thiols: an Alternative Sunthesis of Alkyl-cabamide Coenzyme Analogues. Proc Chem Soc, 1963: p. 211-12. 167. Suto, R.K., N.E. Brasch, O.P. Anderson, and R.G. Finke, Synthesis, characterization, solution stability, and X-ray crystal structure of the thiolatocobalamin gamma- glutamylcysteinylcobalamin, a dipeptide analogue of glutathionylcobalamin: insights into the enhanced Co-S bond stability of the natural product glutathionylcobalamin. Inorg Chem, 2001. 40(12): p. 2686-92. 168. Adler, N., T. Medwick, and T.J. Poznanski, Reaction of hydroxocobalamin with thiols. J Am Chem Soc, 1966. 88(21): p. 5018-20. 169. McCaddon, A. and G. Davies, Co-administration of N-acetylcysteine, vitamin B12 and folate in cognitively impaired hyperhomocysteinaemic patients. Int J Geriatr Psychiatry, 2005. 20(10): p. 998-1000.
153
170. Eikelboom, J.W., E. Lonn, J. Genest, Jr., G. Hankey, and S. Yusuf, Homocyst(e)ine and cardiovascular disease: a critical review of the epidemiologic evidence. Ann Intern Med, 1999. 131(5): p. 363-75. 171. Nygard, O., S.E. Vollset, H. Refsum, L. Brattstrom, and P.M. Ueland, Total homocysteine and cardiovascular disease. J Intern Med, 1999. 246(5): p. 425-54. 172. Lentz, S.R., Mechanisms of homocysteine-induced atherothrombosis. J Thromb Haemost, 2005. 3(8): p. 1646-1654. 173. Wakugawa, Y. and H. Kiyohara, Risk factors for cerebral thrombosis. Clinical Practice (Tokyo, Japan), 2005. 24(5): p. 519-522. 174. Dominguez, R.O., E.R. Marschoff, E.M. Guareschi, A.L. Famulari, M.A. Pagano, and J.A. Serra, Homocysteine, vitamin B 12 and folate in Alzheimer's and vascular dementias: the paradoxical effect of the superimposed type II diabetes mellitus condition. Clin Chim Acta, 2005. 359(1-2): p. 163-70. 175. McCaddon, A., S. Tandy, P. Hudson, R. Gray, G. Davies, D. Hill, and J. Duguid, Absence of macrocytic anaemia in Alzheimer's disease. Clin Lab Haematol, 2004. 26(4): p. 259-63. 176. Refsum, H. and A.D. Smith, Low vitamin B-12 status in confirmed Alzheimer's disease as revealed by serum holotranscobalamin. J Neurol Neurosurg Psychiatry, 2003. 74(7): p. 959-61. 177. McCaddon, A., P. Hudson, G. Davies, A. Hughes, J.H.H. Williams, and C. Wilkinson, Homocysteine and cognitive decline in healthy elderly. Dement Geriatr Cogn Disord, 2001. 12(5): p. 309-313. 178. Ravaglia, G., P. Forti, F. Maioli, M. Martelli, L. Servadei, N. Brunetti, E. Porcellini, and F. Licastro, Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am J Clin Nutr, 2005. 82(3): p. 636-643. 179. Randaccio, L., S. Geremia, M. Stener, D. Toffoli, and E. Zandgrando, Electronic properties of the axial Co-C and Co-S bonds in B12 systems - a density functional study. Eur J Inorg Chem, 2002. 1: p. 93-103. 180. Pratt, J.M., G.S. Hayward, H.A.O. Hill, and R.J.P. Williams, Chemistry of vitamin B12. XII. Coordination of nitrogenous bases. J Chem Soc [Section] A: Inorg Phys Theo, 1971. 1: p. 196-200. 181. Pratt, J.M., Photochemistry, in Inorganic Chemistry of Vitamin B12. 1972, Academic Press: London. p. 256-279. 182. Firth, R., H. Hill, P. JM, R. Thorp, and R. Williams, Chemistry of vitamin B12. XI. Formation constants. J Chem Soc A, 1969. 3: p. 381-6. 183. Thusius, D., Kinetics of Cobalamin Substitution Reactions. J Am Chem Soc, 1971. 93(11): p. 2629--35. 184. Perry, C.B., K.L. Brown, X. Zou, and H.M. Marques, The solution structure of some cobalamins determined by NMR-restrained molecular modelling. J Mol Struct, 2005. 737(2-3): p. 245-258. 185. Marques, H.M., Activation parameters for the reaction of aquocobalamin (vitamin B12a) with small anionic and neutral ligands. J Chem Soc, Dalton Trans, 1991(2): p. 339-41. 186. Perry, C.B., M.A. Fernandes, K.L. Brown, X. Zou, E.J. Valente, and H.M. Marques, Probing the Nature of the Co(III) Ion in Cobalamins - Sectroscopic and Structural Investigations of the Reactions of Aquacobalamin (Vitamin B12a) with Ambident Nucleophiles. Eur J Inorg Chem, 2003: p. 2095-2107.
154
187. Garau, G., S. Geremia, L.G. Marzilli, G. Nardin, L. Randaccio, and G. Tauzher, Crystal chemistry and binding of NO2, SCN and SeCN to Co in cobalamins. Acta Crystallogr B, 2003. 59(Pt 1): p. 51-9. 188. McCaddon, A., P. Hudson, L. Abrahamsson, H. Olofsson, and B. Regland, Analogs, ageing and aberrant assimilation of vitamin B12 in Alzheimer's disease. Dement Geriatr Cogn Disord, 2001. 12(2): p. 133-137. 189. Carmel, R., D.S. Karnaze, and J.M. Weiner, Neurologic abnormalities in cobalamin deficiency are associated with higher cobalamin "analogue" values than are hematologic abnormalities. J Lab Clin Med, 1988. 111(1): p. 57-62. 190. Thorndike, J. and W.S. Beck, Coenzyme function of cobalamin analogs from calf kidney. Blood, 1984. 64(1): p. 91-98. 191. Gimsing, P., Cobalamin forms and analogues in plasma and myeloid cells during chronic myelogenous leukaemia related to clinical condition. Br J Haematol, 1995. 89(4): p. 812- 9. 192. Shaw, S., E. Jayatilleke, S. Meyers, N. Colman, B. Herzlich, and V. Herbert, The ileum is the major site of absorption of vitamin B12 analogues. Am J Gastroenterol, 1989. 84(1): p. 22-6. 193. Djalali, M., J.L. Gueant, D. Lambert, S. el Kholty, M. Saunier, and J.P. Nicolas, High- performance liquid chromatographic separation and dual competitive binding assay of corrinoids in biological material. J Chromatogr, 1990. 529(1): p. 81-91. 194. Buccellato, F.R., L. Foi, D. Veber, G. Pravettoni, and G. Scalabrino, Different uptake of cobalamin (vitamin B12) by astrocytes and oligodendrocytes isolated from rat spinal cord. Glia, 2004. 45(4): p. 406-11. 195. Kolhouse, J.F., C. Utley, S.P. Stabler, and R.H. Allen, Mechanism of conversion of human apo- to holomethionine synthase by various forms of cobalamin. J Biol Chem, 1991. 266(34): p. 23010-5. 196. Quadros, E.V. and D.W. Jacobsen, The dynamics of cobalamin utilization in L-1210 mouse leukemia cells: a model of cellular cobalamin metabolism. Biochim Biophys Acta, 1995. 1244(2-3): p. 395-403. 197. Barker, H.A., R.D. Smyth, H. Weissbach, A. Munch-Petersen, J.I. Toohey, J.N. Ladd, B.E. Volcani, and R.M. Wilson, Assay, purification, and properties of the adenylocobamide coenzyme. J Biol Chem, 1960. 235: p. 181-90. 198. Feldman, K., J. Bobo, S. Ensel, Y. Lee, and P. Weinberg, Template-Controlled ligomerization Support Studies. Template Synthesis and Functionalization. J Org Chem, 1990. 55: p. 474-481. 199. Ellman, G.L., A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys, 1958. 74(2): p. 443-50. 200. Brasch, N.E. and R.G. Finke, A simple, convenient and direct method for assessing the purity of cobalamins. J Inorg Biochem, 1999. 73(4): p. 215-9. 201. Fricke, H., Track effect in iochemistry of aqueous solutions. Ann N Y Acad Sci, 1955. 59(4): p. 567-73. 202. Quadros, E.V., P. Sai, and S.P. Rothenberg, Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system. Blood, 1993. 81(5): p. 1239-45. 203. Rothenberg, S.P. and E.V. Quadros, Quantitative methods for measurement of transcobalamin II. Methods Enzymol, 1997. 281: p. 261-8. 204. Kosower, N.S. and E.M. Kosower, Thiol labeling with bromobimanes. Methods Enzymol, 1987. 143: p. 76-84.
155
205. Fahey, R.C. and G.L. Newton, Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Methods Enzymol, 1987. 143: p. 85-96. 206. Jacobsen, D.W., V.J. Gatautis, R. Green, K. Robinson, S.R. Savon, M. Secic, J. Ji, J.M. Otto, and L.M. Taylor, Jr., Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects. Clin Chem, 1994. 40(6): p. 873-81. 207. Quadros, E.V., D.M. Matthews, I.J. Wise, and J.C. Linnell, Tissue distribution of endogenous cobalamins and other corrins in the rat, cat and guinea pig. Biochim Biophys Acta, 1976. 421(1): p. 141-52. 208. Abel, E.W., J.M. Pratt, R. Whelan, and P.J. Wilkinson, The Mechanism of Oxidation of Vitamin B12r by Oxygen. S Afr J Chem, 1977. 30: p. 1-12. 209. Hohenester, E., C. Kratky, and B. Kraeutler, Low-temperature crystal structure of superoxocobalamin obtained by solid-state oxygenation of the B12 derivative cob(II)alamin. J Am Chem Soc, 1991. 113(12): p. 4523-4530. 210. Alsberg, T., J. Minten, J. Haglund, and M. Tornqvist, Determination of hydroxyalkyl derivatives of cobalamin (vitamin B12) using reversed phase high performance liquid chromatography with electrospray tandem mass spectrometry and ultraviolet diode array detection. Rapid Commun Mass Spectrom, 2001. 15(24): p. 2438-45. 211. Kelleher, B.P., K.G. Walshe, J.M. Scott, and S.D. O'Broin, Microbiological assay for vitamin B12 with use of a colistin-sulfate-resistant organism. Clin Chem, 1987. 33(1): p. 52-4. 212. James, S.J., P. Cutler, S. Melnyk, S. Jernigan, L. Janak, D.W. Gaylor, and J.A. Neubrander, Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. Am J Clin Nutr, 2004. 80(6): p. 1611-7. 213. James, S.J., S. Melnyk, G. Fuchs, T. Reid, S. Jernigan, O. Pavliv, A. Hubanks, and D.W. Gaylor, Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism. Am J Clin Nutr, 2009. 89(1): p. 425-30. 214. Besler, H.T. and S. Comoglu, Lipoprotein oxidation, plasma total antioxidant capacity and homocysteine level in patients with multiple sclerosis. Nutr Neurosci, 2003. 6(3): p. 189-96. 215. Goth, L. and M. Vitai, The effects of hydrogen peroxide promoted by homocysteine and inherited catalase deficiency on human hypocatalasemic patients. Free Radic Biol Med, 2003. 35(8): p. 882-8. 216. Qureshi, G.A., A.A. Qureshi, B.R. Devrajani, M.A. Chippa, and S.A. Syed, Is the deficiency of vitamin B12 related to oxidative stress and neurotoxicity in Parkinson's patients? CNS Neurol Disord Drug Targets, 2008. 7(1): p. 20-7. 217. Herrmann, W., H. Schorr, K. Purschwitz, F. Rassoul, and V. Richter, Total homocysteine, vitamin B(12), and total antioxidant status in vegetarians. Clin Chem, 2001. 47(6): p. 1094-101. 218. Richard, E., A. Jorge-Finnigan, J. Garcia-Villoria, B. Merinero, L.R. Desviat, L. Gort, P. Briones, F. Leal, C. Perez-Cerda, A. Ribes, M. Ugarte, and B. Perez, Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC). Hum Mutat, 2009. 30(11): p. 1558-66. 219. Zhou, J. and R.C. Austin, Contributions of hyperhomocysteinemia to atherosclerosis: Causal relationship and potential mechanisms. Biofactors, 2009. 35(2): p. 120-9.
156
220. Austin, R.C., S.R. Lentz, and G.H. Werstuck, Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease. Cell Death Differ, 2004. 11 Suppl 1: p. S56-64. 221. Koga, T., K. Claycombe, and M. Meydani, Homocysteine increases monocyte and T-cell adhesion to human aortic endothelial cells. Atherosclerosis, 2002. 161(2): p. 365-74. 222. Poddar, R., N. Sivasubramanian, P.M. DiBello, K. Robinson, and D.W. Jacobsen, Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells: implications for vascular disease. Circulation, 2001. 103(22): p. 2717-23. 223. Mudd, S.H., H.L. Levy, and J.P. Kraus, Disorders of Transulfuration, in The Metabolic and Molecular Bases of Inherited Disease., C.R. Scriver, et al., Editors. 2001, McGraw- Hill: New York. 224. Zhang, C., J. Kawauchi, M.T. Adachi, Y. Hashimoto, S. Oshiro, T. Aso, and S. Kitajima, Activation of JNK and transcriptional repressor ATF3/LRF1 through the IRE1/TRAF2 pathway is implicated in human vascular endothelial cell death by homocysteine. Biochem Biophys Res Commun, 2001. 289(3): p. 718-24. 225. Alam, M.M., A.A. Mohammad, U. Shuaib, C. Wang, U. Ghani, B. Schwindt, K.G. Todd, and A. Shuaib, Homocysteine reduces endothelial progenitor cells in stroke patients through apoptosis. J Cereb Blood Flow Metab, 2009. 29(1): p. 157-65. 226. Dong, F., X. Zhang, S.Y. Li, Z. Zhang, Q. Ren, B. Culver, and J. Ren, Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells. Cardiovasc Toxicol, 2005. 5(1): p. 9- 20. 227. Zhang, C., Y. Cai, M.T. Adachi, S. Oshiro, T. Aso, R.J. Kaufman, and S. Kitajima, Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response. J Biol Chem, 2001. 276(38): p. 35867-74. 228. Papatheodorou, L. and N. Weiss, Vascular oxidant stress and inflammation in hyperhomocysteinemia. Antioxid Redox Signal, 2007. 9(11): p. 1941-58. 229. Barbato, J.C., O. Catanescu, K. Murray, P.M. Dibello, and D.W. Jacobsen, Targeting of Metallothionein by L-Homocysteine. A Novel Mechanism for Disruption of Zinc and Redox Homeostasis. Arterioscler Thromb Vasc Biol, 2007. 27(1): p. 49-54. 230. Handy, D.E., Y. Zhang, and J. Loscalzo, Homocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasing translation. J Biol Chem, 2005. 280(16): p. 15518-25. 231. Sawle, P., R. Foresti, C.J. Green, and R. Motterlini, Homocysteine attenuates endothelial haem oxygenase-1 induction by nitric oxide (NO) and hypoxia. FEBS Lett, 2001. 508(3): p. 403-6. 232. Topal, G., A. Brunet, E. Millanvoye, J.L. Boucher, F. Rendu, M.A. Devynck, and M. David-Dufilho, Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin. Free Radic Biol Med, 2004. 36(12): p. 1532-41. 233. Moshal, K.S., N. Metreveli, I. Frank, and S.C. Tyagi, Mitochondrial MMP activation, dysfunction and arrhythmogenesis in hyperhomocysteinemia. Curr Vasc Pharmacol, 2008. 6(2): p. 84-92. 234. Moshal, K.S., S.M. Tipparaju, T.P. Vacek, M. Kumar, M. Singh, I.E. Frank, P.K. Patibandla, N. Tyagi, J. Rai, N. Metreveli, W.E. Rodriguez, M.T. Tseng, and S.C. Tyagi,
157
Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia. Am J Physiol Heart Circ Physiol, 2008. 295(2): p. H890-7. 235. Baydas, G., S.T. Koz, M. Tuzcu, E. Etem, and V.S. Nedzvetsky, Melatonin inhibits oxidative stress and apoptosis in fetal brains of hyperhomocysteinemic rat dams. J Pineal Res, 2007. 43(3): p. 225-31. 236. Olteanu, H. and R. Banerjee, Redundancy in the pathway for redox regulation of mammalian methionine synthase: reductive activation by the dual flavoprotein, novel reductase 1. J Biol Chem, 2003. 278(40): p. 38310-4. 237. Padovani, D. and R. Banerjee, Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone. Biochemistry, 2006. 45(30): p. 9300-6. 238. Green, R. and J.W. Miller, Vitamin B12 deficiency is the dominant nutritional cause of hyperhomocysteinemia in a folic acid-fortified population. Clin Chem Lab Med, 2005. 43(10): p. 1048-51. 239. Hildebrandt, A.G., I. Roots, M. Tjoe, and G. Heinemeyer, Hydrogen peroxide in hepatic microsomes. Methods Enzymol, 1978. 52: p. 342-50. 240. Lang, D., M.B. Kredan, S.J. Moat, S.A. Hussain, C.A. Powell, M.F. Bellamy, H.J. Powers, and M.J. Lewis, Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions. Arterioscler Thromb Vasc Biol, 2000. 20(2): p. 422-7. 241. Ungvari, Z., A. Csiszar, J.G. Edwards, P.M. Kaminski, M.S. Wolin, G. Kaley, and A. Koller, Increased superoxide production in coronary arteries in hyperhomocysteinemia: role of tumor necrosis factor-alpha, NAD(P)H oxidase, and inducible nitric oxide synthase. Arterioscler Thromb Vasc Biol, 2003. 23(3): p. 418-24. 242. Heffelfinger, S.C., H.H. Hawkins, J. Barrish, L. Taylor, and G.J. Darlington, SK HEP-1: a human cell line of endothelial origin. In Vitro Cell Dev Biol, 1992. 28A(2): p. 136-42. 243. Honma, K., M. Kohsaka, N. Fukuda, N. Morita, and S. Honma, Effects of vitamin B12 on plasma melatonin rhythm in humans: increased light sensitivity phase-advances the circadian clock? Experientia, 1992. 48(8): p. 716-20. 244. Sombolos, K., T. Fragia, T. Natse, G. Bartholomatos, A. Karagianni, G. Katsaris, F. Christidou, G. Bamichas, M. Stangou, and N. Papagalanis, The effect of long-term intravenous high dose B-complex vitamins with or without folic acid on serum homocysteine in hemodialysis patients. J Nephrol, 2002. 15(6): p. 671-5. 245. Ballinger, S.W., Mitochondrial dysfunction in cardiovascular disease. Free Radic Biol Med, 2005. 38(10): p. 1278-95. 246. Boveris, A. and A. Navarro, Brain mitochondrial dysfunction in aging. IUBMB Life, 2008. 60(5): p. 308-14. 247. Jou, S.H., N.Y. Chiu, and C.S. Liu, Mitochondrial dysfunction and psychiatric disorders. Chang Gung Med J, 2009. 32(4): p. 370-9. 248. Rezin, G.T., G. Amboni, A.I. Zugno, J. Quevedo, and E.L. Streck, Mitochondrial dysfunction and psychiatric disorders. Neurochem Res, 2009. 34(6): p. 1021-9. 249. Schapira, A.H., Mitochondrial dysfunction in neurodegenerative diseases. Neurochem Res, 2008. 33(12): p. 2502-9. 250. Victor, V.M., N. Apostolova, R. Herance, A. Hernandez-Mijares, and M. Rocha, Oxidative stress and mitochondrial dysfunction in atherosclerosis: mitochondria-targeted antioxidants as potential therapy. Curr Med Chem, 2009. 16(35): p. 4654-67. 251. Victor, V.M., J.V. Espulgues, A. Hernandez-Mijares, and M. Rocha, Oxidative stress and mitochondrial dysfunction in sepsis: a potential therapy with mitochondria-targeted antioxidants. Infect Disord Drug Targets, 2009. 9(4): p. 376-89.
158
252. Allan, L.A. and P.R. Clarke, Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation. Febs J, 2009. 276(21): p. 6063-73. 253. Cocheme, H.M., G.F. Kelso, A.M. James, M.F. Ross, J. Trnka, T. Mahendiran, J. Asin- Cayuela, F.H. Blaikie, A.R. Manas, C.M. Porteous, V.J. Adlam, R.A. Smith, and M.P. Murphy, Mitochondrial targeting of quinones: therapeutic implications. Mitochondrion, 2007. 7 Suppl: p. S94-102. 254. Rocha, M., J.V. Esplugues, A. Hernandez-Mijares, and V.M. Victor, Mitochondrial- targeted antioxidants and oxidative stress: a proteomic prospective study. Curr Pharm Des, 2009. 15(26): p. 3052-62. 255. Tauskela, J.S., MitoQ--a mitochondria-targeted antioxidant. IDrugs, 2007. 10(6): p. 399- 412. 256. Bennett, M.R., Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture. Cardiovasc Res, 1999. 41(2): p. 361-8. 257. Tedgui, A. and Z. Mallat, Apoptosis as a determinant of atherothrombosis. Thromb Haemost, 2001. 86(1): p. 420-6. 258. Kim, D.J., J.M. Koh, O. Lee, N.J. Kim, Y.S. Lee, Y.S. Kim, J.Y. Park, K.U. Lee, and G.S. Kim, Homocysteine enhances apoptosis in human bone marrow stromal cells. Bone, 2006. 39(3): p. 582-90. 259. Wang, H., M. Yoshizumi, K. Lai, J.C. Tsai, M.A. Perrella, E. Haber, and M.E. Lee, Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine. J Biol Chem, 1997. 272(40): p. 25380-5. 260. Lin, C.P., Y.H. Chen, J.W. Chen, H.B. Leu, T.Z. Liu, P.L. Liu, and S.L. Huang, Cholestin (Monascus purpureus rice) inhibits homocysteine-induced reactive oxygen species generation, nuclear factor-kappaB activation, and vascular cell adhesion molecule-1 expression in human aortic endothelial cells. J Biomed Sci, 2008. 15(2): p. 183-96. 261. Mangiarotti, G., C. Canavese, M. Salomone, A. Thea, A. Pacitti, M. Gaido, V. Calitri, D. Pelizza, W. Canavero, and A. Vercellone, Hypervitaminosis B12 in maintenance hemodialysis patients receiving massive supplementation of vitamin B12. Int J Artif Organs, 1986. 9(6): p. 417-20. 262. Elsbach, P. and J. Weiss, Oxygen-dependent and oxygen-independent mechanisms of microbicidal activity of neutrophils. Immunol Lett, 1985. 11(3-4): p. 159-63. 263. Suarez-Moreira, E., J. Yun, C.S. Birch, J.H. Williams, A. McCaddon, and N.E. Brasch, Vitamin B(12) and Redox Homeostasis: Cob(II)alamin Reacts with Superoxide at Rates Approaching Superoxide Dismutase (SOD). J Am Chem Soc, 2009. 264. Earnest, C.P., K.A. Wood, and T.S. Church, Complex multivitamin supplementation improves homocysteine and resistance to LDL-C oxidation. J Am Coll Nutr, 2003. 22(5): p. 400-7. 265. Crocket, J.A., Cyanocobalamin in asthma. Acta Allergol, 1957. 11(4): p. 261-8. 266. Molad, Y., B. Rachmilewitz, Y. Sidi, J. Pinkhas, and A. Weinberger, Serum cobalamin and transcobalamin levels in systemic lupus erythematosus. Am J Med, 1990. 88(2): p. 141-4. 267. Remacha, A.F., M. Montagud, J. Cadafalch, A. Riera, R. Martino, and E. Gimferrer, Vitamin B12 transport proteins in patients with HIV-1 infection and AIDS. Haematologica, 1993. 78(2): p. 84-8. 268. Solomon, L.R., Disorders of cobalamin (Vitamin B12) metabolism: Emerging concepts in pathophysiology, diagnosis and treatment. Blood Rev, 2006.
159
269. Simic, M.G. and M.Z. Hoffman, Addition of Superoxide Radical Anion to Cobalt(II) Macrocyclic Complexes in Aqueous Solution. J Am Chem Soc, 1977. 99(7): p. 2370-1. 270. Schneider, Z. and A. Stroinski, Comprehensive B12, ed. W. deGruyter. 1987, Berlin, New York. 271. McCord, J.M. and I. Fridovich, Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem, 1969. 244(22): p. 6049-55. 272. Butler, J., W.H. Koppenol, and E. Margoliash, Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion. J Biol Chem, 1982. 257(18): p. 10747-50. 273. Nazhat, N.B., B.T. Golding, G.R. Johnson, and P. Jones, Destruction of Vitamin B12 by reaction with ascorbate: the role of hydrogen peroxide and the oxidation state of cobalt. J Inorg Biochem, 1989. 36(2): p. 75-81. 274. Spasojevic, I., I. Batinic-Haberle, R.D. Stevens, P. Hambright, A.N. Thorpe, J. Grodkowski, P. Neta, and I. Fridovich, Manganese(III) biliverdin IX dimethyl ester: a powerful catalytic scavenger of superoxide employing the Mn(III)/Mn(IV) redox couple. Inorg Chem, 2001. 40(4): p. 726-39. 275. Yamada, R., S. Shimizu, and S. Fukui, Disproportionation of vitamin B 12r under various mild conditions. Biochemistry, 1968. 7(5): p. 1713-9. 276. Hohenester, E., C. kratky, and B. Kräutler, Low-Temperature Crystal Structure of Superoxocobalamin obtained by Solid-State Oxygenation of the B12 Derivativa Cob(II)alamin. J Am Chem Soc, 1991. 113: p. 4523-30. 277. Prigoda, S.V. and I.B. Afanas'ev, Interaction of oxygen Anion-Radical O2.- with Hydroxycobalamin and Cyanocobalamin. Koordinatsionnaya Khimiya, 1978. 4(9): p. 1386-90. 278. Mendes, P., GEPASI: a software package for modelling the dynamics, steady states and control of biochemical and other systems. Comput Appl Biosci, 1993. 9(5): p. 563-71. 279. Mendes, P., Biochemistry by numbers: simulation of biochemical pathways with Gepasi 3. Trends Biochem Sci, 1997. 22(9): p. 361-3. 280. Oltean, S. and R. Banerjee, Nutritional modulation of gene expression and homocysteine utilization by vitamin B12. J Biol Chem, 2003. 278(23): p. 20778-84. 281. Day, B.J., M. Patel, L. Calavetta, L.Y. Chang, and J.S. Stamler, A mechanism of paraquat toxicity involving nitric oxide synthase. Proc Natl Acad Sci U S A, 1999. 96(22): p. 12760-5. 282. Mitchell, J.B., A. Samuni, M.C. Krishna, W.G. DeGraff, M.S. Ahn, U. Samuni, and A. Russo, Biologically active metal-independent superoxide dismutase mimics. Biochemistry, 1990. 29(11): p. 2802-7. 283. Weiss, R.H., A.G. Flickinger, W.J. Rivers, M.M. Hardy, K.W. Aston, U.S. Ryan, and D.P. Riley, Evaluation of activity of putative superoxide dismutase mimics. Direct analysis by stopped-flow kinetics. J Biol Chem, 1993. 268(31): p. 23049-54. 284. Nagano, T., T. Hirano, and M. Hirobe, Superoxide dismutase mimics based on iron in vivo. J Biol Chem, 1989. 264(16): p. 9243-9. 285. Pasternack, R.F. and B. Halliwell, Superoxide Dismutase Activities of an iron Porphyrin and Other Iron Complexes. JACS, 1979. 101(4): p. 1026-27. 286. Batinic-Haberle, I., I. Spasojevic, R.D. Stevens, P. Hambright, P. Neta, A. Okado- Matsumoto, and I. Fridovich, New class of potent catalysts of O2.-dismutation. Mn(III) ortho-methoxyethylpyridyl- and di-ortho-methoxyethylimidazolylporphyrins. Dalton Trans, 2004(11): p. 1696-702.
160
287. Batinic-Haberle, I., I. Spasojevic, R.D. Stevens, B. Bondurant, A. Okado-Matsumoto, I. Fridovich, Z. Vujaskovic, and M.W. Dewhirst, New PEG-ylated Mn(III) porphyrins approaching catalytic activity of SOD enzyme. Dalton Trans, 2006(4): p. 617-24. 288. Mc Guire, P.J., A. Parikh, and G.A. Diaz, Profiling of oxidative stress in patients with inborn errors of metabolism. Mol Genet Metab, 2009. 98(1-2): p. 173-80. 289. Scalabrino, G., D. Veber, and E. Mutti, Experimental and clinical evidence of the role of cytokines and growth factors in the pathogenesis of acquired cobalamin-deficient leukoneuropathy. Brain Res Rev, 2008. 59(1): p. 42-54. 290. Tamura, J., K. Kubota, H. Murakami, M. Sawamura, T. Matsushima, T. Tamura, T. Saitoh, H. Kurabayshi, and T. Naruse, Immunomodulation by vitamin B12: augmentation of CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12 treatment. Clin Exp Immunol, 1999. 116(1): p. 28-32. 291. Weinberg, J.B., Y. Chen, N. Jiang, B.E. Beasley, J.C. Salerno, and D.K. Ghosh, Inhibition of nitric oxide synthase by cobalamins and cobinamides. Free Radic Biol Med, 2009. 46(12): p. 1626-32. 292. Tangpong, J., P. Sompol, M. Vore, W. St Clair, D.A. Butterfield, and D.K. St Clair, Tumor necrosis factor alpha-mediated nitric oxide production enhances manganese superoxide dismutase nitration and mitochondrial dysfunction in primary neurons: an insight into the role of glial cells. Neuroscience, 2008. 151(2): p. 622-9. 293. Ai, L., M. Rouhanizadeh, J.C. Wu, W. Takabe, H. Yu, M. Alavi, R. Li, Y. Chu, J. Miller, D.D. Heistad, and T.K. Hsiai, Shear stress influences spatial variations in vascular Mn- SOD expression: implication for LDL nitration. Am J Physiol Cell Physiol, 2008. 294(6): p. C1576-85. 294. Teoh, M.L., W. Sun, B.J. Smith, L.W. Oberley, and J.J. Cullen, Modulation of reactive oxygen species in pancreatic cancer. Clin Cancer Res, 2007. 13(24): p. 7441-50. 295. Huang, T.T., E.J. Carlson, I. Raineri, A.M. Gillespie, H. Kozy, and C.J. Epstein, The use of transgenic and mutant mice to study oxygen free radical metabolism. Ann N Y Acad Sci, 1999. 893: p. 95-112. 296. Willcox, B.J., J.D. Curb, and B.L. Rodriguez, Antioxidants in cardiovascular health and disease: key lessons from epidemiologic studies. Am J Cardiol, 2008. 101(10A): p. 75D- 86D. 297. Zhang, S.H., R.L. Reddick, J.A. Piedrahita, and N. Maeda, Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science, 1992. 258(5081): p. 468-71.