UNIVERSITY OF CALGARY Effect of the human serum albumin concentration

UNIVERSITY OF CALGARY
Effect of the human serum albumin concentration
on the metabolism of cisplatin in vitro
by
Thomas Terry Morris
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF CHEMISTRY
CALGARY, ALBERTA
APRIL, 2014
© Thomas Terry Morris 2014
Abstract
Size-exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic
emission spectrometer (ICP-AES) was employed to study the effect of the human serum albumin
(HSA) concentration on the metabolism of cisplatin (CP) in human blood plasma in vitro.
Plasma from 14 healthy adults and 11 pediatric cancer patients was spiked with CP and the Pt
distribution was determined at the 5 min and 2 hr time point. The results revealed that as the
HSA concentration decreased, the percent of Pt-bound to HSA decreased; whereas the percent of
Pt-hydrolysis products increased, while other Pt-species were largely unaffected. These changes
were corroborated by fortifying the cancer patient plasma with HSA to healthy concentrations
followed by the addition of CP. In summary, these findings imply that the infusion of cancer
patients with HSA to healthy plasma levels may decrease the fraction of free hydrolysis
products, which may alleviate the severe toxic side effects of CP.
ii
Acknowledgements
I would like to express my sincere gratitude to my supervisor Dr. Jürgen Gailer for giving
me the opportunity to work on these fascinating projects. His perpetual enthusiasm,
encouragement, and guidance have inspired me to broaden my knowledge and strengthen my
analytical skills.
I owe my deepest gratitude to Dr. Aru Narendran for his assistance and advice, as well as
for his help with the recruitment of cancer patients. I would also like to thank Karen Mazil for
her constant assistance with the ethics submission for this project, as well as with the recruitment
of the cancer patients.
I wish to express my warm and sincere thanks to Prof. Kevin Thurbide for being in my
supervisory committee and for all of the analytical knowledge he passed on. I am very grateful to
Dr. Roland Roesler for serving on my supervisory committee. It is a pleasure for me to thank Dr.
Ann-Lise Norman for accepting to be the internal examiner, and to Dr. Thomas Baumgartner for
being the neutral chair on my defense committee.
I am grateful to Mandy Chan at the Canadian Sport Centre, University of Calgary for
being so helpful in drawing blood from my volunteers for research. I would like to thank all of
the volunteers for being so generous to donate blood for my research.
Many thanks go to the Department of Chemistry administrative staff, especially to
Bonnie King and Janice Crawford for being so kind in clarifying all my questions. Alberta
Innovates Health Solution is greatly acknowledged for their financial support for my project, as
well as the University of Calgary for their financial support during my degree period.
It was a great pleasure to work with the past and current members of the Gailer group. I
would like to thank Melani and Elham for all of their help, advice, and support they provided
iii
during my degree. I greatly appreciated having them both around, and could not imagine better
group members to share this experience with.
Finally, I would like to convey my deepest gratitude to my parents and sister for their
love and support throughout my life, without whom I would not be where I am today. This
dissertation would not have been possible without all of you.
iv
Dedication
To my loving parents and sister…
v
Table of Contents
Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ iii
Dedication ............................................................................................................................v
Table of Contents ............................................................................................................... vi
List of Tables ................................................................................................................... viii
List of Figures and Illustrations ...........................................................................................x
List of Symbols, Abbreviations and Nomenclature ......................................................... xiii
CHAPTER 1 INTRODUCTION .........................................................................................1
1.1 Cancer as a public health problem .............................................................................1
1.2 Cancer treatment methods .........................................................................................2
1.1.1 Radiation therapy...............................................................................................2
1.1.2 Chemotherapy....................................................................................................2
1.3 Cisplatin (CP) ............................................................................................................3
1.3.1 Discovery ...........................................................................................................3
1.3.2 Mechanism of action .........................................................................................5
1.3.3 Limitations of cisplatin in the treatment of cancer patients ..............................8
1.3.3.1 Adverse toxic side effects ........................................................................8
1.3.3.2 Ameliorating agents that modulate the metabolism of cisplatin..............9
1.3.3.3 Resistance ..............................................................................................10
1.3.4 Strategies to optimize selective toxicity ..........................................................12
1.4 Interactions between metal based drugs and blood constituents .............................14
1.4.1 Human serum albumin (HSA) .........................................................................15
1.4.2 Transferrin (Tf) ................................................................................................18
1.4.3 α2-macroglobulin ............................................................................................18
1.4.4 Hemoglobin (Hb).............................................................................................18
1.5 Influence of plasma protein concentrations on the metabolism/toxicity of medicinal
drugs .......................................................................................................................19
1.6 Determination of metalloproteins in plasma by SEC-ICP-AES ..............................21
1.6.1 Analytical complexity of plasma .....................................................................21
1.6.2 SEC-ICP-AES .................................................................................................21
1.7 Research objectives..................................................................................................23
CHAPTER 2 EXPERIMENTAL.......................................................................................26
2.1 Chemicals and solutions ..........................................................................................26
2.2 SEC-ICP-AES system..............................................................................................26
2.3 Blood collection and plasma analysis by Calgary Laboratory Services (Project 1) 27
2.4 Analysis of CP spiked human plasma......................................................................30
2.4.1 Analysis of plasma from healthy adults ..........................................................30
2.4.2 Analysis of plasma from pediatric cancer patients ..........................................32
2.5 Plasma protein binding of potentially novel anticancer compounds (Project 2) .....33
CHAPTER 3 EFFECT OF THE PLASMA HUMAN SERUM ALBUMIN
CONCENTRATION ON THE METABOLISM OF CISPLATIN IN VITRO........35
vi
3.1 Overview ..................................................................................................................35
3.2 Metabolism of CP in plasma of healthy controls .....................................................35
3.2.1 Quality control of analytical results ................................................................35
3.2.2 Cu-specific chromatograms .............................................................................36
3.2.3 Fe and Zn-specific chromatograms .................................................................38
3.2.4 Pt-specific chromatograms ..............................................................................39
3.3 Metabolism of CP in plasma of pediatric cancer patients .......................................44
3.3.1 Quality control of analytical results ................................................................45
3.3.2 Cu-specific chromatograms .............................................................................46
3.3.3 Fe and Zn-specific chromatograms .................................................................46
3.3.4 Pt-specific chromatograms ..............................................................................47
3.4 Comparison of plasma from healthy adults with pediatric cancer plasma ..............49
3.5 Comparison of pediatric cancer patient plasma with HSA fortified plasma ...........51
CHAPTER 4 PLASMA PROTEIN BINDING OF POTENTIALLY NOVEL METALBASED ANTICANCER COMPOUNDS.................................................................56
4.1 Investigation of 56MESS and 56MESSCu ..............................................................56
4.2 Investigation of DiPy and SOOS .............................................................................59
CHAPTER 5 CONCLUSIONS .........................................................................................61
5.1 Effect of the plasma HSA concentration on the metabolism of CP in vitro ............61
5.2 Plasma protein binding of potentially novel metal-based anticancer compounds ...63
CHAPTER 6 FUTURE WORK ........................................................................................64
CHAPTER 7 REFERENCES ............................................................................................66
APPENDIX A ....................................................................................................................78
APPENDIX B ....................................................................................................................79
APPENDIX C ....................................................................................................................83
APPENDIX D ....................................................................................................................87
APPENDIX E ....................................................................................................................90
APPENDIX F.....................................................................................................................91
APPENDIX G ....................................................................................................................94
APPENDIX H ....................................................................................................................96
APPENDIX I .....................................................................................................................98
vii
List of Tables
Table 1.1: Toxic side effects exerted by CP and carboplatin [74, 75]. ........................................... 9
Table 1.2: Variations of CP binding to HSA following the use of different incubation
conditions and analysis techniques. ...................................................................................... 17
Table 2.1: Reference values for investigated blood plasma parameters provided by CLS [35]. .. 29
Table 3.1: Plasma concentrations of chosen constituents from healthy controls. For
comparison, the reference range pertaining to healthy adults is depicted at the bottom.
Italicized values are outside of the reference range. ............................................................. 36
Table 3.2: Areas of all Pt peaks expressed as % values of total Pt obtained after SEC-ICPAES analysis of human plasma spiked with CP after incubation at 37°C. * Corresponds
to the average of two determinations. ................................................................................... 42
Table 3.3: A more detailed analysis of the Pt peak areas and retention times observed at the 2
hr time point expressed as % values of total Pt obtained after SEC-ICP-AES analysis of
human plasma spiked with CP. * Corresponds to one analysis. **Corresponds to the
average of two analyses. The average difference between the analyses for PP-3 was
0.7% PP = Protein bound Pt-species, HP = Pt-containing hydrolysis product. .................... 43
Table 3.4: Concentration of HSA, Tf, creatinine, and B.U.N. in blood plasma of pediatric
cancer patients compared to healthy levels. The ranges pertaining to healthy children are
depicted at the bottom of the table. ....................................................................................... 45
Table 3.5: Peak areas of all Pt peaks expressed as % values of total Pt obtained after SECICP-AES analysis of human plasma spiked with CP after incubation at 37°C. ................... 47
Table 3.6: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of human plasma spiked with CP after incubation at 37°C for 2 hr. PP = Protein
bound Pt-species, PH = Pt-containing hydrolysis product.................................................... 49
Table 3.7: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from healthy adults (Avg. 42 g HSA/L) and pediatric cancer patients
(Avg. 26 g HSA/L) spiked with CP after incubation at 37°C for 2 hr. PP = Protein
bound Pt-species, HP = Pt-containing hydrolysis product, H = Healthy adults, C =
Pediatric cancer patients........................................................................................................ 50
Table 3.8: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from pediatric cancer patients (Avg. 26 g HSA/L) and the HSA
fortified plasma spiked with CP after incubation at 37°C for 2 hr. PP = Protein bound
Pt-species, HP = Pt-containing hydrolysis product, H = Healthy adults, C = Pediatric
cancer patients. ...................................................................................................................... 52
viii
Table 3.9: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from C-11 after being spiked with CP and varying amounts of HSA.
PP = Protein bound Pt-species, HP = Pt-containing hydrolysis product. ............................. 54
Table 3.10: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICPAES analysis of plasma from C-02 after being spiked with CP and varying amounts of
HSA. PP = Protein bound Pt-species, HP = Pt-containing hydrolysis product. ................... 54
Table 3.11: Linear regression analysis showing the increase of Pt-bound HSA per gram of
HSA added to plasma. ........................................................................................................... 55
ix
List of Figures and Illustrations
Figure 1.1: Methyl-bis(beta-chloroethyl)amine hydrochloride – one of the mustard gases
used to treat lymphsarcoma [20]. In the form of its hydrochloride salt, it can be readily
dissolved in saline for intravenous administration [18]. ......................................................... 3
Figure 1.2: Structure of Pt-based anti-cancer drugs that are (A) in worldwide clinical use [34]
and (B) approved for use in Asia. ........................................................................................... 4
Figure 1.3: Schematic illustration of the hydrolysis of CP in aqueous solution [41, 44]. .............. 5
Figure 1.4: Schematic depiction of the intracellular fate of CP in mammalian cells.
Abbreviations: MRP2 – multidrug resistance-associated protein 2, Ctr1 – copper
transport protein 1, OCT2 – organic cation transport protein 2. GSH-Pt complex
obtained in cell-free systems as well as extracted from L1210 murine leukemia cells, as
determined by mass spectroscopic analysis [43]. ................................................................... 6
Figure 1.5: Different types of DNA-CP adducts. Y=H2O/OH, Cl, or protein [34].
Republished with permission from Elsevier. .......................................................................... 7
Figure 1.6: Ameliorating agents co-administered with CP used to reduce nephrotoxicity. ........... 9
Figure 1.7: Schematic representation of the main biochemical mechanisms that are involved
in the development of resistance of a tumor cell to CP treatment. ....................................... 10
Figure 1.8: Schematic of tumor targeting by nanohybrids via the EPR effect [119].
Reproduced with permission from Elsevier. ......................................................................... 14
Figure 1.9: Conceptual depiction of the biochemistry of CP after it is introduced into the
mammalian bloodstream. Abbreviations: BP – binding protein, Ctr1 – copper transport
protein, OCT2 – organic cation transport protein 2 [128]. ................................................... 15
Figure 1.10: Medicinal drugs used for the treatment of anxiety. .................................................. 20
Figure 1.11: Novel potential anticancer compounds for plasma protein binding investigation.
(A) 56MESS, (B) 56MESSCu, (C) [u[2-[2-(2-mercapto-ethoxy)-ethoxy]ethanethiol]]bis(2,2':6',2''-terpyridine)platinum(II) [{Pt(terpy)} 2(SOOS)], which will be
referred to as SOOS, and (D) [u-4,4′-dipyridine bis(2,2':6',2''-terpyridine)platinum(II)]
[{Pt(terpy)}2(DiPy)], which will be referred to as DiPy. ..................................................... 25
Figure 2.1: C and Fe specific chromatograms obtained for plasma from healthy adults after
the addition of CP after 5 min (H-02 no peak splitting, H-16 HSA & Tf peak splitting)..... 31
Figure 3.1: SEC-ICP-AES derived C-specific chromatograms obtained for the analysis of 14
plasma samples from healthy controls spiked with CP (0.04 mg/mL). The mixtures were
incubated at 37 °C and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200
10/300 GL column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS
x
buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. Detector: ICP-AES
at 193.091 nm (C). (A) No splitting of peaks corresponding to HSA, (B) peak splitting
of HSA peak. The retention times of the molecular weight markers are depicted on top. ... 37
Figure 3.2: SEC-ICP-AES derived Cu-specific chromatograms for the analysis of 14 plasma
samples from healthy controls spiked with CP (0.04 mg/mL). The mixtures were
incubated at 37 °C and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200
10/300 GL column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS
buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. Detector: ICP-AES
at 324.754 nm (Cu). The retention times of the molecular markers are depicted on top.
The most intense ceruloplasmin peak was observed after the ICP torch was exchanged
and shifted. ............................................................................................................................ 38
Figure 3.3: Representative SEC-ICP-AES derived Cu-specific chromatograms for the
analysis of H-12-2 spiked with CP (0.04 mg/mL). The mixture was incubated at 37 °C
and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column
(30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow
rate: 1.0 mL/min. Injection volume: 500 µL. Detector: ICP-AES at 324.754 nm (Cu).
The retention times of the molecular markers are depicted on top. ...................................... 39
Figure 3.4: Representative Pt-specific chromatograms obtained for the analysis of H-17-1
spiked with CP (0.04 mg/mL). The mixture was incubated at 37 °C and analyzed after 5
min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13
µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 214.423 nm (Pt). Peaks 1-3: protein
bound Pt-species, peaks 4-5: Pt-containing hydrolysis products, peak 6: CP. The
retention times of the molecular weight markers are depicted on top. ................................. 41
Figure 3.5: HSA concentrations plotted against their corresponding Pt peak areas of Pt-bound
HSA expressed as a percentage. ........................................................................................... 44
Figure 3.6: Representative SEC-ICP-AES derived Cu-specific chromatograms for the
analysis of 11 cancer patients spiked with CP (0.04 mg/mL). The mixture was incubated
at 37 °C and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL
column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH
7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. Detector: ICP-AES at 324.754
nm (Cu). The retention times of the molecular weight markers are depicted on top. .......... 46
Figure 3.7: Representative Pt-specific chromatograms obtained for the analysis of C-11
spiked with CP (0.04 mg/mL). The mixture was incubated at 37 °C and analyzed after 5
min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13
µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 214.423 nm (Pt). The retention times
of the molecular weight markers are depicted on top. .......................................................... 48
Figure 3.8: Proposed model to link the observed in vitro observed results with (A) high HSA
concentrations in healthy controls and the (B) lower concentrations of HSA in pediatric
xi
cancer patients with the increase in severe toxic side effects. The font size corresponds
to the relative concentration of metabolites or plasma proteins in plasma. .......................... 51
Figure 3.9: HSA concentrations of pediatric cancer patients as well as their plasma fortified
with HSA (36 g HSA/L and 42 g HSA/L) plotted against their corresponding Pt peak
areas of Pt-bound HSA expressed as a %. ............................................................................ 53
Figure 4.1: Representative C, Fe, and Pt-specific chromatograms obtained for the analysis of
rabbit plasma spiked with 56MESS (100 µmol/L). The mixture was incubated at 37 °C
and samples were analyzed after 5 min and 1 hr . Stationary phase: Superdex 200
10/300 GL column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS
buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. The retention times
of the molecular markers are depicted on top. ...................................................................... 57
Figure 4.2: Representative C, Fe, and Cu-specific chromatograms obtained for the analysis of
rabbit plasma spiked with 56MESSCu (100 µmol/L). The mixture was incubated at 37
°C and samples were analyzed after 5 min and 1 hr . Stationary phase: Superdex 200
10/300 GL column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS
buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. The retention times
of the molecular markers are depicted on top. ...................................................................... 58
Figure 4.3: Representative C, Fe, and Pt-specific chromatograms obtained for the analysis of
rabbit plasma spiked with DiPy (100 µmol/L). The mixture was incubated at 37 °C and
samples were analyzed after 5 min and 1 hr . Stationary phase: Superdex 200 10/300 GL
column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH
7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. The retention times of the
molecular markers are depicted on top. ................................................................................ 59
Figure 4.4: Representative C, Fe, and Pt-specific chromatograms obtained for the analysis of
rabbit plasma spiked with SOOS. The mixture was incubated at 37 °C and samples were
analyzed after 4 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d.,
13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0
mL/min. Injection volume: 500 µL. The retention times of the molecular markers are
depicted on top. ..................................................................................................................... 60
xii
List of Symbols, Abbreviations and Nomenclature
Symbol
A
AES
ATP7A/ATP7B
B.U.N.
BP
CE
CLS
CNS
CP
CTR1
CuZnSOD
EPR
G
GFR
GSH
GSTπ
Hb
HP
HPLC
HSA
ICP
MRP
MS
NAC
NER
OCT
PBS
PP
SEC
STS
Tf
Definition
Adenine
Atomic emission spectroscopy
Copper transporting ATPase 1&2
Blood urea nitrogen
Binding protein
Capillary electrophoresis
Calgary Laboratory Services
Central nervous system
Cisplatin
Copper transport protein
Copper-zinc superoxide dismutase
Enhanced permeability and retention effect
Guanine
Glomular filtration rate
Glutathione
Glutathione-S-transferase-π
Hemoglobin
Hydrolysis Products
High performance liquid chromatography
Human serum albumin
Inductively coupled plasma
Multidrug resistance protein
Mass spectrometry
N-acetyl cysteine
Nucleotide excision repair
Organic cation transport protein
Phosphate buffered saline
Plasma protein
Size exclusion chromatography
Sodium thiosulfate
Transferrin
xiii
CHAPTER 1 INTRODUCTION
1.1 Cancer as a public health problem
All mammalian organisms are made up of ~200 cell types [1], all of which grow and
divide in a controlled way to replenish those that have become non-viable. Aged or damaged
cells are removed from the organism by a process known as apoptosis, and are replaced with new
cells. When the genetic material of a cell is damaged by mutations of the DNA, it can adversely
affect cell growth and division, and result in their uncontrolled proliferation. The plethora of
diseases in which abnormal cells divide without control is referred to as cancer [2], with more
than 75,000 people expected to die from the disease each year in Canada alone [3]. This
corresponds to cancer being the primary cause of all deaths in Canada (~30%) [4], and
approximately half of all men and one third of women will develop cancer in their lifetime [5].
Currently there are ~100 different types of cancer, typically named for the organ or cell
type in which the disease started (e.g., leukemia, lymphoma, and head and neck cancers) [2]. The
most frequent types of cancers are lung, colorectal, breast, pancreas and prostate cancer, with
lung cancer alone expected to cause 20,100 deaths/year in Canada [3]. Among males, the three
most prevalent cancers are prostate (43%), colorectal (9%), and melanoma of the skin (7%),
whereas the most common ones among females are breast (41%), uterus (8%), and colorectal
cancer (8%) [5]. Cancer cells can spread from their point of origin to other parts of the body via
the bloodstream and lymph system by a process referred to as metastasis. Metastasis is the last
stage of cancer, and is the hardest to treat since every cancer cell must be eradicated from the
body to achieve remission, making it extremely difficult to accomplish.
1
In terms of cancer treatments, only a few methods are available, including surgery,
radiation therapy, and chemotherapy, but promising new approaches such as immunotherapy and
stem cell therapy are also currently being explored.
1.2 Cancer treatment methods
1.1.1 Radiation therapy
Radiation therapy employs the utilization of X-rays to kill cancer cells. The generated
free oxygen radicals will damage vital biochemical molecules, such as DNA, and eventually kill
the irradiated cell. While radiation therapy can eradicate cancer, it may sometimes just slow its
growth [6]. However, better results are being achieved through advances in radiation equipment
[7], and optimizing the radiation schedule [8]. Although radiation therapy has curative or
palliative potential in roughly half of all incident solid tumors [9], its side effects, such as
mucosal and skin damage, can be quite severe [10]. In addition, fundamental activities of daily
life, such as speech, breathing, eating, and drinking may also be negatively affected [11, 12].
1.1.2 Chemotherapy
The term “chemotherapy” was coined in the early 1900’s by German chemist Paul
Ehrlich who began to develop drugs to treat bacterial diseases and defined it as the use of
chemicals to treat illnesses [13]. At the time, he referred to these chemicals as ‘magic bullets’,
which were intended to bind to receptors on the surface of disease causing bacteria and kill the
latter, therefore achieving selective toxicity. The development of chemotherapy for the treatment
of cancer began in the mid 1940’s [14] and involved observations made during World War I and
II. People who had died because of exposure to mustard gases displayed atrophy of their bone
2
marrow and their lymph nodes, but not of other cell types [15, 16]. These findings suggested that
mustard gas (Figure 1.1) appeared to selectively affect only certain populations of cells [17, 18],
and led to the testing of mustard gases in animal tumors (e.g. lymphosarcoma in mice) [17-20].
These promising results led to its testing in a human patient, which resulted in a significant
regression of the tumor mass over two weeks [21]. These strongly encouraging findings resulted
in the discovery of other families of antitumor drugs, such as anticancer antibiotics (e.g.
bleomycin), antimitotics, and cisplatin in 1969 [14]. Currently, there are over 100 drugs that are
being used for cancer chemotherapy [6], and each of these is typically used in combination with
other drugs/therapies to shrink tumors before and/or after surgery or radiation therapy.
Figure 1.1: Methyl-bis(beta-chloroethyl)amine hydrochloride – one of the mustard gases used to
treat lymphsarcoma [20]. In the form of its hydrochloride salt, it can be readily dissolved in
saline for intravenous administration [18].
1.3 Cisplatin (CP)
1.3.1 Discovery
The serendipitous discovery of the antineoplastic (inhibiting or preventing the
proliferation of malignant cells) activity of this metal-based drug emerged from a seemingly
unrelated experiment on the effects of an electric field on the cell division of Escherichia coli
[22-24]. Using Pt electrodes in a buffer solution, researchers observed that the bacteria extruded
3
long filaments, and stopped replicating. More detailed analysis revealed that the cell division
cycle was inhibited by Pt-amine complexes that had formed near the surface of Pt electrodes, and
eventually resulted in the development of CP (Figure 1.2) as an anticancer drug [22]. After its
approval for clinical use by the Food and Drug Administration in 1978, the intravenous
administration with CP was discovered to be particularly useful for the treatment of testicular
and ovarian cancers, due to their inherent sensitivity to CP treatment [25-27]. The overall cure
rate exceeds 90% for testicular cancer, and is nearly 100% if this type of cancer is detected early
[28]. Treatment with CP is also a more common treatment option against other types of solid
tumors including head/neck, lung, cervical, bladder, and breast cancer [29-32]. Today, Pt-based
anti-cancer therapeutics are among the most frequently used chemotherapeutic agents employed
to treat cancer patients worldwide (Figure 1.2), with CP still holding the status of being one of
the world’s best-selling anti-cancer drugs [33].
Figure 1.2: Structure of Pt-based anti-cancer drugs that are (A) in worldwide clinical use [34]
and (B) approved for use in Asia.
4
1.3.2 Mechanism of action
Ever since the discovery of the anticancer activity of CP, major efforts have been devoted
to elucidate the biochemical mechanisms of its antitumor activity in order to possibly design
novel Pt based drugs with superior pharmacological profiles. Once intravenously administered,
the comparatively high chloride blood plasma concentration of ~105 mM [35] greatly diminishes
the hydrolysis of CP. Its translocation from the bloodstream into cells is driven by passive
diffusion, and/or active transport mediated by copper transport protein 1 (CTR1) or organic
cation transporters (OCT) [36-38]. The comparatively low intracellular [Cl-] of ~4-20 mM [39,
40] allows CP to undergo hydrolysis reactions to form a variety of positively charged hydrolysis
products (Figure 1.3) [41]. It is the latter that are capable of reacting with numerous intracellular
biomolecules, such as the tri-peptide, glutathione (GSH) (Figure 1.4) [42, 43], as well as reacting
mono and bi-functionally with the negatively charged DNA (Figure 1.4 & 1.5) [34].
Figure 1.3: Schematic illustration of the hydrolysis of CP in aqueous solution [41, 44].
5
Figure 1.4: Schematic depiction of the intracellular fate of CP in mammalian cells.
Abbreviations: MRP2 – multidrug resistance-associated protein 2, Ctr1 – copper transport
protein 1, OCT2 – organic cation transport protein 2. GSH-Pt complex obtained in cell-free
systems as well as extracted from L1210 murine leukemia cells, as determined by mass
spectroscopic analysis [43].
6
Figure 1.5: Different types of DNA-CP adducts. Y=H2O/OH, Cl, or protein [34]. Reproduced
from Pizarro, A.M.. Unusual DNA binding modes for metal anticancer complexes.Biochemie;
91(10): 1198-1211. Copyright © 2009. Published by Elsevier Masson SAS. All rights reserved.
The antitumor activity of CP is believed to be due to its capability to form bi-functional
DNA cross-links. The N7 atom of guanine - the preferred target due to it having the highest
electron density of all four nucleobases [45] - and adenine located in the major groove of the
double helix are the most accessible and reactive nucleophilic sites for the coordination of Pt to
DNA [46]. The predominant adducts that are formed between CP hydrolysis products and DNA
are the intrastrand crosslinks 1,2-d(GG) (65%), 1,2-d(AG) (25%), and 1,3-d(GG) (5-10%), as
well as a small percentage of interstrand crosslinks and mono-functional adducts (Figure 1.5)
[47-55]. Although it has not been definitively proven, it is generally believed that the 1,27
intrastrand DNA adduct, which bends the double helix towards the major groove is responsible
for apoptosis of the cell. This premise is based on the discovery of a group of chromosomal
proteins that are involved in the regulation of DNA-dependent processes, such as transcription,
replication, recombination and DNA repair [56]. These proteins specifically recognize this type
of Pt-DNA adduct and inhibit the repair of 1,2-intrastrand crosslinks that were formed by CP
[57]. Further support in favour of this hypothesis came from the finding that nucleotide excision
repair, an important DNA repair system, repairs 1,3-intrastrand adducts with higher efficiency
than the 1,2-instrastrand adducts [58]. However, it is generally thought that the kink in the
overall DNA structure forces the individual strands to remain together when replication requires
that they must be unravelled. Since cellular processes like transcription and replication are
adversely affected, the result is cellular death by apoptosis or necrosis [59] [60].
Transplatin has substantially lower cytotoxicity than CP [61, 62]. Since both isomers bind
to DNA almost exclusively by coordination to the N7 nitrogen atom of purine bases (higher
affinity for guanine over adenine) [63, 64], the lack of cytotoxicity of transplatin must be related
to the different type of DNA adducts that are formed by this isomer. This may be attributed to
transplatin lacking the ability to form 1,2-intrastrand crosslinks with DNA due to steric
constraints, which are the most abundant adducts formed by CP [65]. Indeed, transplatin mainly
forms 1,3-intrastrand and interstrand cross-links [66].
1.3.3 Limitations of cisplatin in the treatment of cancer patients
1.3.3.1 Adverse toxic side effects
The intravenous administration of patients with CP is associated with severe, doselimiting toxic side effects, including nephrotoxicity and ototoxicity (Table 1.1) [67]. Because of
8
this, research efforts are aimed at synthesizing advanced / 3rd generation metal complexes for
chemotherapy which exert a higher selective toxicity, and should therefore diminish the severe
toxic side effects [34]. However, of the ~3000 tested newly synthesized Pt compounds [68], only
a fraction (about 30) have entered clinical trials, and merely three Pt drugs have been approved
worldwide (Figure 1.2A) [69, 70], with nedaplatin, lobaplatin, and heptaplatin being approved in
China, South Korea, and Japan (Figure 1.2B) [70-73].
Table 1.1: Toxic side effects exerted by CP and carboplatin [74, 75].
Cisplatin
Carboplatin
Nephrotoxicity
Myelosuppression
Peripheral Neuropathy (50%)
Peripheral Neuropathy (4-6%)
Ototoxicity (31%)
Ototoxicity (1.1%)
Gastrointestinal Toxicity
1.3.3.2 Ameliorating agents that modulate the metabolism of cisplatin
The toxic side effects of CP, however, have also been ameliorated in model organisms by
the co-administration with sodium thiosulfate (STS) [42], N-acetylcysteine (NAC) (Figure 1.6)
[76, 77], and D-methionine [78]. When STS is incubated in plasma with CP, a biologically
inactive Pt-STS complex is formed which provides a feasible bio-molecular mechanism by
which the toxic side effects of CP could be reduced by this ameliorating agent in vivo [79].
Figure 1.6: Ameliorating agents co-administered with CP used to reduce nephrotoxicity.
9
1.3.3.3 Resistance
Although CP is widely used to treat cancer patients, its efficacy is limited to certain types
of cancers, and the development of resistance during the treatment is a frequently encountered
problem [34]. This phenomenon is generally considered to be multifactorial, and may be
attributed to: (i) reduced drug accumulation & enhanced cell efflux, (ii) intracellular inactivation
by thiol containing species (e.g. glutathione), and (iii) increased repair of Pt-DNA adducts
(Figure 1.7) [46]. These resistance mechanisms have been demonstrated to be cell line-dependent
so that a particular tumor may exhibit one, two, or even all of these resistance mechanisms [80,
81].
Figure 1.7: Schematic representation of the main biochemical mechanisms that are involved in
the development of resistance of a tumor cell to CP treatment.
10
Reduced drug accumulation of CP has been observed in a variety of CP-resistant cell
lines, and is the principal mechanism of resistance [82]. Previous studies have shown that Ctr1, a
copper transport protein, is regulated by extracellular Cu+ concentration; high extracellular Cu+
triggers internalization and degradation of Ctr1 [83, 84]. Since Ctr1 is involved in CP uptake, the
internalization of Ctr1 has been reproduced in human ovarian cancer cells with CP within
minutes [85, 86], and has also been shown to occur in yeast cells [36].
Enhanced cell efflux of Pt species by molecular pumps in response to CP treatment has
been demonstrated in several studies, and revealed the multidrug resistance protein (MRP)
family to play a major role. This family of proteins is composed of at least seven members
(MRP1-7), all of which are ATP-dependent transmembrane proteins [87]. Several of these MRPs
are associated with the cellular efflux of a variety of drugs [88]. However, only MRP1 and
MRP2 appear to be of significance in some CP-resistant tumor models [89]. MRP2 seems to
have a more significant effect on the efflux of Pt species, since it is associated with an increased
expression in CP resistant cells [90-92]. In the past few years, an additional family of protein
efflux pumps has been linked to CP resistance. ATP7A and ATP7B are two copper-transporting
proteins which have been shown to be overexpressed in CP resistant tumor cells [93, 94]. It is
thought that these two copper transporting proteins initiate the efflux of CP and/or its hydrolysis
products from the Golgi apparatus directly for elimination from the cell [82] [95]. This reduces
the amount of CP interacting with DNA, in turn reducing its cytotoxicity.
With regard to the cytosolic fate of CP, aquation reactions inside the cell lead to the
formation of hydrolysis products which can interact with other nucleophilic components inside
the cell, such as GSH. These side reactions inactivate CP, and reduce the availability of the drug
to form the intended DNA adducts. Additionally, several clinical studies have demonstrated a
11
correlation between increased GSH synthesis and CP resistance [96, 97]. Although the reaction
between GSH and CP’s hydrolysis products can occur spontaneously in the cytosol (Figure 1.4),
it can also be catalyzed by the enzyme GSH-S-transferase-π (GSTπ), which may be up-regulated
in CP resistant cells [98]. An involvement of this enzyme in CP resistance is further supported by
studies documenting less favorable responses to treatment with CP for cases with elevated
expressions of GSTπ, such as a high GSTπ expression found in 87.5% of patients without
clinical response to chemotherapy [99]. This effect results in shorter overall survival times and
progression-free time with respect to patients with lower levels of GSTπ [100-103].
Increased repair of Pt DNA adducts is another important mechanism of resistance, having
already been demonstrated in several murine and human tumor cell lines [82]. Excision of the Pt
DNA adducts is associated with the nucleotide excision repair (NER) pathway, a complex
process involving ~17 different proteins [104-106]. Cells deficient in NER show extreme
sensitivity to Pt damage [107]. Likewise, increased expression of the ERCC1 gene, an essential
gene in the NER pathway, directly correlates with increased resistance to Pt therapy in ovarian
cancer, gastric cancer, colorectal cancer, and lung cancer [107].
1.3.4 Strategies to optimize selective toxicity
Since CP is associated with severe toxic side effects, alternative approaches are
continuously being developed to diminish the side effects in patients. These include, but are not
limited to:
1. The tethering of cell-targeting moieties to CP allows for an increase in cellular targeting,
while also reducing toxic side effects. One such molecule is folic acid. Folate receptors
are overexpressed in cancer cells, allowing for the selective uptake of folic acid [108,
12
109]. In ovarian, uterus, brain, kidney, head and neck, mesothelium, and endometrium
tumors, the receptors are overexpressed by up to two orders of magnitude [110-114]. The
receptors on tumor cells are expressed on the cells that are in contact with circulating
blood [115].
2. Use of liposomes to exploit the enhanced permeability and retention (EPR) effect: The
EPR effect describes how certain sizes of molecules tend to accumulate in cancerous
tissue much more than they do in normal tissues due to the gaps (200 to 2000 nm) in the
endothelial tumor tissue (Figure 1.8) [116-118]. By exploiting the differences between
normal and cancerous tissues, it allows for improved targeting and delivery of anticancer
drugs to improve efficacy and potentially reduce toxic side-effects. This effect is evident
in a large number of tumor types, and is used as a strategy for the creation of novel
cancer-specific drugs that are comprised of but not limited to micelles (1:10 CP to lipid
ratio) and nanocapsules (10:1 CP to lipid ratio) [118-120]. The nanoscale size of these
carriers is important, since it prevents them from entering normal tissues or removal by
renal clearance. As a result, these drugs have greater exposure to the tumor sites
compared to the low molecular weight drugs, which can be rapidly cleared from
circulation [120]. One of the most promising and highly developed drug carriers currently
used are liposomes (phospholipid bilayer vesicles) [121]. Liposomal delivery can
improve the amount of drug available to the tumor cell by increasing the vascular
permeability and therefore the influx and retention of the drug [122]. Liposomes have
many advantages such as biocompatibility, versatility (allowing the existence of
biologically active drugs either within the hydrophobic phospholipid bilayers or within
the hydrophilic aqueous cavity), and the potential to shield drugs from inactivation before
13
delivery to the target site (such as plasma proteins deactivating CP) [122]. The
physicochemical properties of the liposome can be varied by manipulation of size, charge
and surface properties during formulation [123]. A liposomal formulation of doxorubicin,
an anticancer drug, is already in use in the clinic [124].
Figure 1.8: Schematic of tumor targeting by nanohybrids via the EPR effect [118]. Reproduced
from Prakash, S. Polymeric nanohybrids and functionalized carbon nanotubes as drug delivery
carriers for cancer chemotherapy. Adv. Drug. Deliv. Rev; 63(14-15): 1340-1351. Copyright ©
2011. Published by Elsevier Masson SAS. All rights reserved.
1.4 Interactions between metal based drugs and blood constituents
Since CP is intravenously administered to patients, its interaction with all blood
constituents must be considered. CP is known to be absorbed by erythrocytes, where it fragments
hemoglobin (Hb) [125], and its hydrolysis products interact with plasma proteins [46]. In fact,
the half-life of CP in blood plasma after the treatment of adults is approximately five months, but
several factors can affect this (e.g. age, glomular filtration rate) [126]. The interactions of CP
14
hydrolysis products with plasma proteins play a key role not only in the context of its therapeutic
effects, but also in mediating its toxic side effects [67]. It is currently unclear how many plasma
proteins are involved (Figure 1.9) and whether the associated interactions are reversible or
irreversible.
Figure 1.9: Conceptual depiction of the biochemistry of CP after it is introduced into the
mammalian bloodstream. Abbreviations: BP – binding protein, Ctr1 – copper transport protein,
OCT2 – organic cation transport protein 2 [127].
1.4.1 Human serum albumin (HSA)
In humans, HSA (66.3 kDa) is the most abundant protein in blood plasma [40-45 g/L
(0.6 mM)] [45, 128]. One of its main functions is the transport of fatty acids, bilirubin, essential
metal ions, steroid hormones, vitamins, and pharmaceuticals, including metallodrugs, to organs
[129-131]. HSA also performs a variety of other physiologically important functions, such as
15
controlling osmotic blood pressure [129, 132, 133]. In vitro studies have shown that 3 hr after CP
was added to human plasma, ~50% of the Pt was bound to HSA [127], mainly via Cys-34 and
Met-298 [67, 134]. A pharmacologically relevant dose of CP was added to bovine plasma and
incubated at 37°C for 45.5 hr and after 48 hr of dialysis there was only an approximate loss of
1% of Pt that was once bound to BSA [135]. These results suggest that the binding of CPhydrolysis products to HSA is predominantly irreversible. Controversy surrounds the role that
HSA-bound Pt plays in the context of exerting its desired antitumor activity of CP as well as its
unintended toxic side effects.
CP derived Pt-species that were bound to HSA have been demonstrated to be
therapeutically inactive in a human ovarian cancer cell line [136]. In addition, various studies
with human cell lines, including the human epidermoid carcinoma A431 cell line, have shown
cytotoxic effects only at comparatively high concentrations of a largely uncharacterized synthetic
HSA-CP adduct [IC50 (72 hr) of 85 µM vs 0.61 µM of free CP] [137, 138]. In contrast, a study
involving animals implanted with tumors has shown HSA-bound Pt to have equivalent activity
compared to free CP at equal dosages [139]. Related to this, hypoalbuminemic patients
responded poorly to CP treatment [140-142], and HSA-CP complexes exerted cytotoxicity to
cancer cells [139, 143]. In support of these findings, infusion of a preformed HSA-CP complex
significantly increased patient survival times compared to untreated patients (median survival
time of 109 days compared to 56 days) [139, 143] and revealed an even higher Pt tumor
concentrations after the administration of a synthetic HSA-CP adduct to cancer patients
compared to free CP at the same dosage [143]. Thus, the antitumor activity of intravenous
administration of CP appears to be determined in vivo by both free CP as well as HSA-CP
adducts that are formed in the bloodstream.
16
With regard to the toxic side effects of CP hydrolysis products bound to HSA, there is a
similar ambiguity. Compared to conventional CP therapy, for example, the administration of
HSA-CP complexes to patients has resulted in fewer toxic side effects, such as nephrotoxicity
and ototoxicity at the same dose [143-145], while increasing the tumor Pt concentrations [143].
The parameters which govern the HSA-CP adduct formation in vitro are not fully
understood either. It can be influenced by the molar ratio between the drug and the protein, initial
concentration of the protein, incubation time, and pH of the solution (Table 1.2). For example,
HSA was shown to bind as much as 20 mol of CP after 14 days of incubation at 37°C when a 60fold drug excess over the protein was used [146]. These studies, however, only examined the
binding capacity in vitro, and not the cytotoxicity of the formed complex. Thus, it is unclear if
different Pt-bound HSA complexes exert different antitumor activities, especially in vivo.
Table 1.2: Variations of CP binding to HSA following the use of different incubation conditions
and analysis techniques.
Method
Drug
Concentration
(M)
5 x 10-5 to 6 x
10-4
Protein
Incubation
Concentration Conditions
(M)
1 x 10-5
pH 7.4, 100 mM
Gel-filtration[146]
NaCl, 37°C, 48photometry
144 hr
-3
-5
8 x 10
pH 7.4, 37°C, 48
Gel filtration- 1.6 x 10
hr
ICP-AES[147]
-5
-5
[148]
5 x 10 to 1 x 5 x 10
pH 7.4, 100 mM
CE-ICP-MS
10-3
NaCl, 37°C, 48 hr
-5
-5
[149]
1 x 10 to 1 x 5 x 10
pH 7.4, 100 mM
CE
10-3
NaCl, 37°C, 48 hr
17
Drug
Molecules
/ Protein
3.5-20.0
5.1
0.7-10.2
6.3
1.4.2 Transferrin (Tf)
Tf (79 kDa [150]) is found in human blood plasma at a concentration of ~2.5 g /L (35
µM) [151] and serves as a Fe3+ transporter, for which it has two binding sites. CP derived
hydrolysis products are capable of binding to Tf, competing directly with Fe3+ at Thr-457,
located in the C-lobe of Tf [152, 153]. Additionally, CP-derived hydrolysis products will bind to
O donors (Tyr136, Tyr317) and S donors (Met256, His273, His578) on the surface of both apoand holo-Tf, [150]. CP derived hydrolysis products bound to holo-Tf do not interfere with the
recognition of this protein by the Tf receptor, and can therefore enter the cell at a similar rate as
holo-Tf [150]. However, the conjugation of CP derived hydrolysis products to holo-Tf greatly
reduced the amount of platinated DNA adducts compared to free CP in the human breast cancer
cell line MCF-7, as CP was unable to be released from the protein intracellularly, thus leading to
reduced cytotoxicity compared to CP [150].
1.4.3 α2-macroglobulin
α2-macroglobulin (820 kDa, 2.5-4 g/L ) [154] has also been shown to play a role in the
pharmacokinetics of CP [155]. For example, 0.14 µM α2-macroglobulin once incubated with 1.7
mM CP for 4 hr, 95% of the CP-derived Pt was bound to the protein [155]. There are a limited
number of studies that have been performed to investigate the interaction of CP with this protein
in detail.
1.4.4 Hemoglobin (Hb)
Hb (65 kDa, 120-170 g/L) [156], the Fe containing protein which is responsible for the
transportation of O2 by its heme group, can also form adducts with CP derived hydrolysis
18
products [125]. After the incubation of Hb (typically 2 mM in red blood cells [157]) in whole
blood from healthy human volunteers with CP at a clinically relevant concentration (0.05 µM)
and a higher concentration (10 µM) for 24 hr, the heme group was cleaved from the protein,
leaving only CP-Hb complexes and no free drug [125, 158, 159].
1.5 Influence of plasma protein concentrations on the metabolism/toxicity of medicinal
drugs
Plasma protein concentrations can greatly influence the distribution, the metabolism, and
the excretion of medicinal drugs, leading to variances in their pharmacokinetics which may
directly affect the toxic side effects [160]. An example that illustrates how HSA may potentially
play a role in mediating toxic side effects of medicinal drugs was uncovered for methadone, a
drug which is used to relieve pain [160]. Using human plasma in vitro, the % binding of
methadone was investigated as a function of various HSA plasma concentrations (between 4 g
HSA/L – 50 g HSA/L). The study revealed that increasing HSA concentrations in plasma
increased the percent of methadone bound (~8% at 4 g HSA/L and ~44% at 50 g HSA/L), while
only changing the drug concentration and leaving the HSA constant had little effect on the % of
methadone that was bound to HSA.
Another study investigated the effect of varying the HSA concentration on the
pharmacokinetics of chlordiazepoxide and diazepam (used to treat anxiety) and their undesired
effects (CNS depression) in vivo (Figure 1.10) [161]. Of 1037 patients receiving diazepam, 9.3%
of patients with hypoalbuminemia (less than 30 g HSA/L) had CNS depression, while this was
only seen in 2.9% of patients with normal serum HSA levels (≥ 40 g HSA/L). Similar trends
were observed with chloriazepoxide. These results clearly demonstrate that the plasma
19
concentration of HSA can directly affect the pharmacokinetics and the toxicity of the drug in
vivo. Since HSA represents 50% of the total protein content in human blood plasma (healthy
adults) [162] and it has been demonstrated to interact with CP-derived hydrolysis products, its
plasma concentration will significantly affect the pharmacokinetics of CP.
Figure 1.10: Medicinal drugs used for the treatment of anxiety.
Since cancer patients have much lower blood plasma concentration of HSA due to
impaired renal and/or liver function and a general lack of appetite [163], it is important to
understand the interaction between pharmacological relevant doses of CP and varying HSA
plasma concentrations as this could pave the way to improve treatment methods to reduce the
toxic side effects.
20
1.6 Determination of metalloproteins in plasma by SEC-ICP-AES
1.6.1 Analytical complexity of plasma
From an analytical point of view, the separation of plasma proteins represents a herculean
task since plasma contains between 3700-10000 proteins [164-166]. In addition, the
concentrations of individual plasma proteins can vary up to 10 orders of magnitude [167]. A
variety of instrumental analytical techniques have therefore been developed to address the
complexity that is associated with the analysis of plasma. Conceptually, one can either attempt to
analyze the entire plasma proteome (2D gel electrophoresis) [165], or one can simplify the
problem by analyzing an inherently less complex subproteome. The mammalian plasma
metalloproteome represents such a subproteome [168, 169]. The most abundant 22 proteins
represent 99% of the total protein content of plasma [170], and since ~half of these contain metal
cofactors, the analysis of metalloproteins greatly simplifies the analysis as well as provides
valuable information. The most frequently used instrumental approaches for the analysis of
plasma for the contained metalloproteins are hyphenated techniques, which are based on a
separation method and a high sensitivity method for metal detection and quantification [171].
One such technique is size-exclusion chromatography (SEC) coupled on-line to an inductively
coupled plasma atomic-emission spectrometer (ICP-AES), which was developed by Shawn
Manley et al. [172].
1.6.2 SEC-ICP-AES
SEC-ICP-AES is a comparatively inexpensive [compared to SEC-ICP mass spectrometry
(MS)], multi-element detection approach that one can employ to determine the plasma
distribution of metals from metalloproteins, such as Fe, Cu, and Zn in less than 25 min. The use
21
of phosphate buffered saline (PBS) as the mobile phase allows one to maintain conditions as
close as possible to physiological as the mobile phase buffer can adversely affect the stability of
metal-protein bonds during analysis. This can result in a re-distribution of zinc among various
plasma proteins during analysis [173]. With this in mind, two main applications of this approach
can be envisioned.
The first application is the use of this hyphenated technique as a diagnostic tool to test for
human disease by detecting increased or decreased concentrations of plasma metalloproteins
[174]. Since the analysis only takes 25 min, changes in the concentrations of the target
metalloproteins can be monitored, while information on other metalloproteins can be
simultaneously obtained. Since CP rapidly hydrolyses in plasma and interacts with proteins such
as HSA, the distribution of Pt among plasma proteins can be examined in 40 min intervals.
The second application involves probing the bio-inorganic chemistry of environmentally
abundant toxic metals (e.g. Cd) [174] with various blood constituents in the mammalian
bloodstream to better understand their toxicity. Recently, our research group investigated the
plasma protein binding of arsenobetaine in human and rabbit plasma [175]. Through the use of
this technique, it was revealed that arsenobetaine does not bind to plasma proteins > 300 Da over
a 6 hr period. Hence, this study provided direct experimental evidence that metallodrug-plasma
protein interactions which are amendable with this technique can explain the rapid clearance of
arsenobutaine in urine. Subsequently, this technique was applied to study the comparative
hydrolysis and plasma protein binding of CP and carboplatin in human plasma [127] using this
technique. Most recently, this methodology was applied to gain insight into the molecular basis
of how the “ameliorating agents” of STS, NAC, and D-methionine in human plasma in vitro.
22
SEC-ICP-MS is a related methodology that can be employed to investigate the
aforementioned. Due to the much lower detection limits of ICP-MS (0.01-0.1 ppt for Pt)
compared to ICP-AES (20 ppb for Pt) [176], it offers some advantages. However, its
incompatibility to accept PBS buffer due to its high salt content is a major disadvantage that
would render this technique incapable of mimicking physiological conditions. Therefore, SECICP-AES was the method of choice for the study of the metabolism of CP in human plasma in
vitro.
1.7 Research objectives
The interactions of CP with plasma proteins have in general only been investigated under
non-physiological conditions (with regard to the mobile phase) with ICP-MS [60, 177-179]. This
thesis marks the first time that the effect of the HSA concentration on the metabolism of CP in
vitro has been investigated under physiological conditions.
Effect of the plasma HSA concentration on the metabolism of CP in vitro (Chapter 3):
Since HSA plays a key role in the pharmacokinetics of any medicinal drug, as well as CP
[67], varying blood plasma concentrations of this protein may greatly affect the metabolism and
therefore the distribution of Pt to potential target organs. Therefore, the plasma HSA
concentration may play a more important role in mediating the toxic side effects on healthy cells
as well as their effect on cancer cells. The objective of this project was to systematically evaluate
the effect of varying HSA plasma concentrations on the metabolism of CP. A pharmacologically
relevant dose of CP was added to blood plasma of twenty healthy adults (33 – 50 g HSA/L) and
eleven cancer patients (8 – 34 g HSA/L) and the Pt distribution of the obtained mixture was
23
determined after 5 min and 2 hr. In addition, HSA was directly added to the plasma of cancer
patients to be able to investigate the effect of increasing HSA concentration on the metabolism of
CP.
Plasma protein binding of potentially novel metal-based anticancer compounds (Chapter
4):
In collaboration with Dr. Janice Aldrich-Wright (University of Western Sydney), four
potential novel anticancer compounds (Figure 1.11), including 56MESS and 56MESSCu, were
examined with regard to their binding to plasma proteins using rabbit plasma. [(5,6-dimethyl1,10-phenanthroline)(1S-2S-diaminocyclohexane)platinum(II)]2+, also referred to as 56MESS
(Figure 1.11), has been demonstrated to exhibit a significantly lower LC 50 than CP in L1210
cells (mouse lymphocytic leukemia cell line), while also displaying activity in CP resistant cell
lines [180]. The structurally related compound 56MESSCu in which the Pt atom is replaced by
Cu has also been demonstrated to exert anticancer activity in L1210 cells (LC50 0.42 µM) [181].
Pharmacologically relevant concentrations of each compound were added to rabbit plasma, and
the latter was analyzed by SEC-ICP-AES over a 2 hr period to examine their comparative
hydrolysis and plasma protein binding.
24
Figure 1.11: Novel potential anticancer compounds for plasma protein binding investigation.
(A)
56MESS,
(B)
56MESSCu,
(C)
[u[2-[2-(2-mercapto-ethoxy)-ethoxy]ethanethiol]]bis(2,2':6',2''-terpyridine)platinum(II) [{Pt(terpy)}2(SOOS)], which will be referred
to
as
SOOS,
and
(D)
[u-4,4′-dipyridine
bis(2,2':6',2''-terpyridine)platinum(II)]
[{Pt(terpy)}2(DiPy)], which will be referred to as DiPy.
25
CHAPTER 2 EXPERIMENTAL
2.1 Chemicals and solutions
Blue dextran (M.W. 2 000 000 g/mol) and phosphate buffered saline tablets (PBS, 0.01 M
phosphate, 0.0027 M KCl, 0.137 M NaCl) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Cisplatin (1 mg cis-Pt(NH3)2Cl2/mL; this solution also contained 1 mg mannitol and 9 mg
NaCl / mL; sterile) was obtained from Hospira (Montreal, QC, Canada) and carboplatin (10 mg
cis-Pt(NH3)2C4H6(COO)2/mL) from Mayne Pharma (Montreal, QC, Canada). HSA (12.5 g in 50
mL of buffered diluent, stabilized with 0.02 M sodium caprylate and 0.02 M sodium
acetyltryptophanate) was obtained from CSL Behring AG (Bern, Switzerland). PBS buffer (10
mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4) was prepared by dissolving PBS tablets in
the appropriate volume of water (followed by pH adjustment with dilute HCl) and filtration
through 0.45 µm Nylon filter membranes (Mandel Scientific Company Inc., Guelph, ON,
Canada) before use. A mixture of protein standards for SEC column calibration was obtained
from Bio-Rad Laboratories (Hercules, CA, USA) and contained thyroglobulin (670 kDa), ɣglobulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa). All
solutions were prepared with water from a Simplicity water purification system (Millipore,
Billerica, MA, USA). The novel anticancer compounds were obtained from Dr. Janice AldrichWright from the University of Western Sydney, Australia.
2.2 SEC-ICP-AES system
The SEC-ICP-AES system was comprised of a Smartline 1000 HPLC pump (Knauer,
Berlin, Germany) and a Rheodyne 9010 PEEK injection valve (Rheodyne, Rhonert Park, CA,
USA) which was equipped with a PEEK injection loop (0.5 mL). A pre-packed SuperdexTM 200
26
10/300 GL TricornTM high performance size-exclusion chromatography column (30 x 1.0 cm
I.D., 13 µm particle size, separates globular proteins between ~600 and ~10 kDa; GE Healthcare,
Piscataway, NJ, USA) was used for the separation of plasma proteins using PBS-buffer as the
mobile phase at a flow rate of 1.0 mL/min (column temperature 22°C) [174]. Simultaneous
multielement-specific detection of C (193.091 nm), S (180.731 nm), P (213.618 nm), Cu
(324.754 nm), Fe (259.940 nm), Zn (213.856 nm) and Pt (214.423 nm) was achieved with a
Prodigy, high-dispersion, radial-view ICP-AES (Teledyne Leeman Labs, Hudson, NH, USA) at
an Ar gas-flow rate of 19 L/min, an RF power of 1.3 kW, and a nebulizer gas pressure of 35 psi.
A 6.0 min delay was implemented between injection and data acquisition based on the void
volume (440 s/ 7.33 mL) that was determined by the injection of blue dextran and C-specific
detection. The SEC column was calibrated using a mixture of protein standards and the retention
time obtained for each protein standard is indicated on top of each chromatogram along with the
corresponding molecular weight. The data acquisition windows were 1500 s for plasma spiked
with cisplatin and 1140 s for carboplatin. The obtained raw data were imported into SigmaPlot
12 software and smoothed using the bisquare algorithm. Baseline correction and integration of
peak areas were performed with OriginPro 9.1.
2.3 Blood collection and plasma analysis by Calgary Laboratory Services (Project 1)
The collection of blood from humans was approved by the Calgary Conjoint Health
Research Ethics Board (Ethics I.D. E-25315, see Appendix A). Twenty healthy adults (12 males
and 8 females) were recruited for analysis (Adult consent form, see Appendix B). Since it is
logistically and ethically difficult to collect blood from healthy children (age 2-18) which would
have been our first choice for a control group, blood plasma from healthy adults was used as a
27
control group instead. Approximately 12 mL of blood was collected by a certified nurse from the
Department of Kinesiology at the University of Calgary. However, some adults were not able to
donate the full 12 mL for various reasons. After centrifugation at 1000 g (4°C) for 10 min, the
supernatant plasma was removed using a micropipette and pooled. 1.6 mL aliquots of this
homogenous plasma stock were transferred to cyrovials and stored in liquid nitrogen. This size
of aliquot was used to allow for 2 sample injections per vial (800 µL per injection to allow for
rinsing of the 500 µL sample loop). In order to ensure that the total time from blood collection to
sample storage was less than an hr; samples were collected on four different days to allow for an
adequate amount of time to centrifuge the blood and aliquot the plasma.
Eleven pediatric cancer patients were also recruited to the study (Parental consent form,
see Appendix C & Assent form, see Appendix D). Only 11 cancer patients could be recruited
from the Alberta Children’s Hospital over an 8 month period due to time constraints and based
on various inclusion and exclusion criteria (see Appendix E). A one-time donation of ~12 mL of
blood was taken and deposited into two 6 mL heparinized trace metal testing blood collection
tubes (Greiner-Bio-One VacuetteTM, NC, USA). Even though 12 mL of blood does not produce
enough plasma to do triplicate studies, taking a low amount was necessary, since the amount of
blood that can be collected from a child is dependent on their body weight. The blood was then
centrifuged to produce plasma, which was then aliquoted into cryovials and kept frozen until
analysis.
Blood plasma was analyzed by Calgary Laboratory Services (CLS) using the following
techniques (Services Agreement for CLS - Appendix F):
28
 Albumin: Determined by using a colorimetric assay. Knowing the HSA concentration is
essential for determining a relationship between HSA concentrations and the Pt
distribution.
 Transferrin: Determined by using an immunoturbidimetric assay. Tf plays an important
role in the pharmacokinetics of CP [182].
 Creatinine: Determined by using a colorimetric assay. This parameter was analyzed
because it is an important indicator of renal health.
 Blood Urea Nitrogen (B.U.N.): Determined by using a kinetic UV assay. Its levels are
related to the hydration status, as well as renal health of plasma.
Any data obtained from a control blood sample with abnormal levels of these compounds (except
albumin) was excluded from analysis (Table 2.1).
Table 2.1: Reference values for investigated blood plasma parameters provided by CLS [35].
Compound
Healthy
Gender/Age
Reference Range
Albumin
33-48 g/L
Males & Females > 1 year old
Transferrin
2.00-3.60 g/L
Males & Females all ages
Creatinine
50-120 µmol/L
Males > 15 years old
35-100 µmol/L
Females > 15 years old
3.0-7.5 mmol/L
Males 15-54 years old
2.0-7.0 mmol/L
Females 15-54 years old
B.U.N.
One way to assess a patient’s nutritional status is through the measurement of the HSA
[183], as well as the Tf concentration in plasma [184, 185]. The plasma HSA concentration is a
proxy for the rate of protein synthesis and thus overall health [163]. Depending on the stage of
29
the cancer, patients will generally have lower levels of HSA, and in the later stages of cancer,
malnutrition and inflammation can reduce the synthesis of albumin in the liver [186] due to the
increased production of cytokines (small proteins involved in cell signaling), as part of the
inflammatory response to the tumor [186-188]. Therefore, the plasma albumin levels won’t drop
in early stages of cancer, but as the disease progresses, the levels will drop significantly [186,
189]. In addition, a study which investigated the effect of HSA levels in 180 breast cancer
patients found that normal levels of HSA (> 35 g HSA/L) reduced the risk of death by 72%, and
that low levels of HSA adversely affected survival by a statistically significant level for all stages
of the disease [190]. The hydration state can also obscure the effects of a patient’s nutritional
status [191]. B.U.N. levels increase with dehydration (reabsorption), and levels can also build up
if kidney function is impaired. Creatinine is another reliable indicator of kidney function, as poor
clearance will cause a build-up. Typically the B.U.N. to creatinine weight ratio provides more
precise information about dehydration. Ratios greater than 20:1 of B.U.N. to creatinine suggest
dehydration. Since one of CP’s toxic side effects is renal damage, chemotherapy as a treatment
option is limited if creatinine levels are high. However, a decreased dietary intake, as well as
decreasing muscle mass, will reduce levels of creatinine.
2.4 Analysis of CP spiked human plasma
2.4.1 Analysis of plasma from healthy adults
Plasma (1.6 mL) was thawed at room temperature for 45 min and incubated in closed
cryovials at 37°C while gently shaken (125 rpm) for 30 min before CP (67 µL, 0.04 mg/mL) was
added. The obtained mixture was incubated at 37°C for 5 min and then 0.5 mL were withdrawn
for analysis by SEC-ICP-AES. Another aliquot was analyzed 2 hr after CP had been added to
30
plasma. All experiments were carried out in duplicate since the amount of plasma that could be
obtained was limited. With this setup, the 5 min time point served as a control since almost all of
the CP existed as the free drug, whereas the 2 hr time point reflected the metabolism of CP in
plasma. If less than 1.6 mL of plasma was available, only the 2 hr time point was analyzed. The
C, Cu, Fe, and Zn specific chromatograms were simultaneously obtained. C-specific
chromatograms which displayed peak splitting (800-1200 s range) were not included in data
analysis (Figure 2.1). The abrupt loss of the ICP-AES plasma during analysis and software
crashes also meant no results were obtained for some analyses. Thus, 5 controls were not used in
the discussion.
100
40000
PBS-H-02 5 min Carbon
PBS-H-02 5 min Iron
80
Transferrin
HSA
Fe Intensity (CPS)
C Intensity (CPS)
30000
20000
10000
60
40
20
Ferritin
V0
V0
0
0
40000
100
PBS-H-16 5 min Carbon
PBS-H-16 2 5 min Iron
HSA
Transferrin
80
Fe Intensity (CPS)
C Intensity (CPS)
30000
20000
10000
60
40
Ferritin
20
V0
V0
0
0
400
600
800 1000 1200 1400 1600 1800 2000
400
600
800 1000 1200 1400 1600 1800 2000
Retention Time (s)
Retention Time (s)
Figure 2.1: C and Fe specific chromatograms obtained for plasma from healthy adults after the
addition of CP after 5 min (H-02 no peak splitting, H-16 HSA & Tf peak splitting).
31
2.4.2 Analysis of plasma from pediatric cancer patients
Plasma (1.6 mL) was thawed at room temperature for 45 min, incubated at 37°C for 30
min, and analyzed 5 min and 2 hr after CP addition. In contrast to the control plasma analyses,
different amounts of highly pure HSA were added to establish the effect that an increased HSA
concentration has on the metabolism of CP in each investigated plasma sample. The addition of
HSA was thought to increase the percentage of protein bound Pt-species, due to an increase in
the Pt-HSA peak. This would enable us to compare these results to those from the control
samples, as well as potentially see the expected stepwise change in the distribution of Pt.
 Vial 1: 67 µL of CP stock solution was added to plasma (final concentration of 0.04
mg/mL).
 Vial 2: HSA was added to plasma to increase the final HSA concentration to 36 g
HSA/L; then 67 µL of CP stock solution was added (final concentration of 0.04 mg/mL).
 Vial 3: HSA was added to plasma to increase the final HSA concentration to 42 g
HSA/L; then 67 µL of CP stock solution was added (final concentration of 0.04 mg/mL).
In cases that HSA was added to plasma, the CP addition had to be slightly adjusted so the
dosage would remain the same. Since the amount of plasma in the vials from the cancer patients
varied, the volumes of CP and HSA stock solutions added to the plasma needed to be adjusted.
Thus, these two formulas with the CP volume as well as the HSA stock solution volume to be
added were used to calculate the precise amounts needed to acquire proper dosages:
1. (67 µL CP) / (1667 µL total solution) = (Volume of CP added) / (Volume of plasma +
Volume of CP added + Volume of HSA added)
32
2. (Volume of plasma x [HSA] in plasma) + (250 g HSA/L x Volume of HSA stock
solution added) = (Volume of plasma + Volume of CP added + Volume of HSA
added) x Desired [HSA]
When a limited amount of blood plasma was obtained, only the 2 hr time point with a 36
g/L concentration of HSA was determined. Compared to the other time points with the addition
of no HSA and 42 g HSA/L, the 36 g HSA/L time point is in the middle, and thus the least
amount of information would be obtained from this experiment in particular. Therefore, if there
was a low amount of blood plasma from the patient, this experiment was excluded.
The obtained mixtures were incubated at 37°C for 5 min and then 0.5 mL were
immediately injected onto the SEC-ICP-AES system. Another injection was performed 2 hr after
the addition of CP/HSA. A solution containing carboplatin in PBS buffer at equal Pt
concentrations compared to the CP spiked plasma was injected at the beginning and end of each
day to assess the integrity of the SEC column and to calculate the % recovery of Pt from plasma
spiked with CP. A % recovery of 97 ± 14 was calculated. Due to time constraints and eligibility
criteria (see Appendix E), only 11 cancer patients could be recruited from the Alberta Children’s
Hospital over an 8 month period. Different SEC columns were used for the healthy adults and
cancer patients due to the development of a gap in the SEC column for the healthy adults.
2.5 Plasma protein binding of potentially novel anticancer compounds (Project 2)
After thawing rabbit plasma for 45 min at room temperature, it was spiked with 10-50 µL
(concentration dependent) of a solution containing the dissolved compound in PBS buffer to
achieve a final concentration of 100 µmol/L, which is similar to concentrations previously
investigated with the A549 cell line (human lung carcinoma) (150-750 µmol/L) [192].The pH
33
was adjusted to ~7.40 using dilute HCl or NaOH. Three replicates were performed with each
compound, with injection times of 5 min and 1 hr after the compound was added to plasma.
During this time, the plasma was kept in an incubator at 37°C. The SEC column was calibrated
using a mixture of protein standards and the retention time obtained for each protein standard is
indicated at the top of each chromatogram along with the corresponding molecular weight. The
same parameters were used for the SEC-ICP-AES system as described in section 2.2 for project
1.
34
CHAPTER 3 EFFECT OF THE PLASMA HUMAN SERUM ALBUMIN
CONCENTRATION ON THE METABOLISM OF CISPLATIN IN VITRO
3.1 Overview
The goal of this project was to investigate the metabolism of CP in plasma in vitro in 20
healthy adult volunteers and compare them to that in 11 cancer patients. The results of these
studies were intended to provide insight into the development of treatment protocols which may
possibly reduce the toxic side effects of CP in cancer patients.
3.2 Metabolism of CP in plasma of healthy controls
Blood was collected from 20 adults and assigned ID #s (privacy protection). Plasma was
analyzed for creatinine, B.U.N., Tf, and HSA (Table 3.1). Almost all concentrations were within
the reference range for healthy adults. Because the creatinine and B.U.N plasma concentrations
of H-09 were outside the reference range, the result of its analysis was excluded from the
discussion. Since the plasma of the ICP extinguished during the analyses of CP-spiked plasma
for the 2 hr time points of samples H-01, H-03, and H-10, no results could be obtained.
3.2.1 Quality control of analytical results
Based on the relative C-peak intensities of all 4 detected peaks in plasma (Figure 3.1A)
compared to previous studies [174], exclusion criteria to reject results from the discussion were
defined. Since the most intense C-peak corresponds to HSA, the overall shape of this peak was
used as a proxy to guage the quality of the chromatographic separation for all investigated
plasma samples. If peak splitting was observed, the obtained results were excluded from the
discussion. This occured with sample H-18 (Figure 3.1B) and H-20. Therefore, only 14 of the
35
collected blood plasma samples provided results that were used for comparison with the results
from the plasma from the pediatric cancer patients.
Table 3.1: Plasma concentrations of chosen constituents from healthy controls. For comparison,
the reference range pertaining to healthy adults is depicted at the bottom. Italicized values are
outside of the reference range.
ID #
Gender
H-01
Male
H-02
H-03
H-04
H-05
H-06
H-07
H-08
H-09
H-10
H-11
H-12
H-13
H-14
H-15
H-16
H-17
H-18
H-19
H-20
Male
Female
Male
Female
Male
Male
Female
Female
Male
Female
Female
Male
Male
Female
Male
Male
Female
Male
Male
Reference
range
HSA
(g/L)
43
Tf
(g/L)
3.15
Creatinine
(µmol/L)
83
B.U.N.
(mmol/L)
5.0
B.U.N. :
Creatinine
14.9
43
44
46
35
40
41
40
33
45
44
44
41
50
39
43
40
38
44
38
33-48
2.61
2.74
3.10
3.39
2.81
3.04
2.69
3.30
2.72
2.64
2.99
2.48
2.60
3.25
2.70
2.96
3.56
2.67
2.52
2.003.60
111
67
95
61
72
81
75
31
72
74
81
78
95
78
68
73
65
99
88
50-120
(Males)
35-100
(Females)
4.1
3.3
3.9
4.6
4.2
5.4
3.0
1.9
5.8
3.9
4.2
3.8
5.6
4.6
6.6
5.1
3.6
7.5
5.1
3.0-7.5
(Males)
2.0-7.0
(Females)
9.1
12.2
10.2
18.7
14.4
16.
9.9
15.2
19.9
13.1
12.8
12.1
14.6
14.6
24.0
17.3
13.7
18.8
14.4
> 20 =
Dehydratio
n
3.2.2 Cu-specific chromatograms
When fresh rabbit plasma was analyzed previously, 5 Cu-containing entities, namely
coagulation factor V, transcuprein, ceruloplasmin, albumin bound Cu, and small molecular
36
670 kDa
158 kDa
44 kDa 17 kDa
670 kDa
1.35 kDa
158 kDa
44 kDa 17 kDa
1.35 kDa
40000
C Intensity (CPS)
A
n=14
5 min
HSA
B
H-18-1-5min
HSA
30000
20000
V0
V0
10000
0
400
600
800
1000
1200
1400 400
Retention Time (s)
600
800
1000
1200
1400
Retention Time (s)
Figure 3.1: SEC-ICP-AES derived C-specific chromatograms obtained for the analysis of 14
plasma samples from healthy controls spiked with CP (0.04 mg/mL). The mixtures were
incubated at 37 °C and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL
column (30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow
rate: 1.0 mL/min. Injection volume: 500 µL. Detector: ICP-AES at 193.091 nm (C). (A) No
splitting of peaks corresponding to HSA, (B) peak splitting of HSA peak. The retention times of
the molecular weight markers are depicted on top.
weight Cu were detected [174]. Some of these Cu-moieties (coagulation factor V and
transcuprein), however, could not be detected at the 1 hr time point due to the instability of the
involved Cu-protein bonds at room temperature [174]. Since in the present study the control
plasma samples were kept stored frozen until analysis, we did not expect to detect 5 Cu peaks.
Since in ceruloplasmin, all 6 Cu atoms are firmly protein bound [193], this plasma
metalloprotein served as an internal standard, and produced a distinct Cu signal for all 14 plasma
samples from healthy adults at both time points. All retention times of the Cu peaks
corresponding to this metalloprotein were highly reproducible which indicates that data
acquisition was carried out diligently for all samples (Figure 3.2). The reproducibility is further
37
illustrated by plotting the results that were obtained for the analysis of an individual plasma
sample after incubation for 5 min and 2 hr (Figure 3.3). Based on these results, it can be inferred
that all simultaneously obtained Pt-specific chromatograms are reliable.
670 kDa
44 kDa 17 kDa
158 kDa
670 kDa
1.35 kDa
1200
Ce ruloplas m in
5 min
n=14
44 kDa 17 kDa
158 kDa
1.35 kDa
Ce ruloplas m in
2 hr
n=14
Cu Intensity (CPS)
1000
800
600
400
Album in
bound Cu
Album in
bound Cu
200
V0
Sm all m ole cular
w e ight Cu
V0
Sm all m ole cular
w e ight Cu
0
400
600
800
1000
1200
1400 400
Retention Time (s)
600
800
1000
1200
1400
Retention Time (s)
Figure 3.2: SEC-ICP-AES derived Cu-specific chromatograms for the analysis of 14 plasma
samples from healthy controls spiked with CP (0.04 mg/mL). The mixtures were incubated at 37
°C and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column
(30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0
mL/min. Injection volume: 500 µL. Detector: ICP-AES at 324.754 nm (Cu). The retention times
of the molecular markers are depicted on top. The most intense ceruloplasmin peak was observed
after the ICP torch was exchanged and shifted.
3.2.3 Fe and Zn-specific chromatograms
Since the Fe and Zn specific chromatograms that were obtained after the addition of CP
to plasma were very similar between the 5 min and the 2 hr time point, they are presented in
Appendix G & H.
38
670 kDa
158 kDa
44 kDa 17 kDa
1.35 kDa
800
Ceruloplasmin
H-12-2
5 min
2 hr
Cu Intensity (CPS)
600
400
Albumin
bound Cu
200
V0
Small molecular
weight Cu
0
400
600
800
1000
1200
1400
Retention Time (s)
Figure 3.3: Representative SEC-ICP-AES derived Cu-specific chromatograms for the analysis
of H-12-2 spiked with CP (0.04 mg/mL). The mixture was incubated at 37 °C and analyzed after
5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13 µm
particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection
volume: 500 µL. Detector: ICP-AES at 324.754 nm (Cu). The retention times of the molecular
markers are depicted on top.
3.2.4 Pt-specific chromatograms
In principle, three separate events occur after CP is injected into the bloodstream; (a)
hydrolysis, (b) the binding of CP hydrolysis products to plasma proteins, and (c) the uptake of
CP and/or its hydrolysis products (free or protein bound) into cells [127]. The hydrolysis of CP
results in the formation of the aqua adducts [PtClOH2(NH3)2]+ and [Pt(OH2)2(NH3)2]2+ [75], as
well as other Pt-containing compounds, such as dimer and trimer complexes [194-196]. These
hydrolysis products can then bind to plasma proteins, including HSA. Thus, the analysis of blood
plasma by SEC-ICP-AES allows one to probe the metabolism of CP by determining the size
39
distribution of Pt species at a given time point. The Pt distribution (protein bound Pt, Pthydrolysis products, and free drug) that was obtained at the 5 min and the 2 hr time point (Figure
3.4) for all 14 plasma samples from healthy controls are summarized in Table 3.2, and will be
discussed first. A more detailed view of all detected Pt peaks at the 2 hr time point is displayed in
Table 3.3 and will be discussed thereafter.
No protein bound Pt was detected 5 min after the addition of a pharmacologically
relevant dose of CP, 2.4 ± 0.3% of CP eluted in the form of hydrolysis products, and 97.6%
remained as the free drug. These findings are in contrast to a previous study which reported that
5 min after spiking human plasma with CP [127], 6.5% of Pt eluted as Pt-bound proteins, 4.6 ±
0.4% as Pt-hydrolysis products, and 89 ± 0.6% as CP for males (n = 3). Similar results were
reported for females (n = 3). In the present study and at the 2 hr time point, 53.0 ± 2.8% of the Pt
was protein bound, while 15.9 ± 1.7% of CP eluted as hydrolysis products, and 31.1 ± 1.7% as
the free drug. Previous studies which determined the Pt distribution after the addition of the same
dose of CP to human plasma at the 3 hr time point were in general agreement with the results
obtained in the present study. For example, the relative peak intensities of all protein bound Pt
peaks and that of the 2 hydrolysis products are very similar to results that were previously
obtained at the 3 hr time point. [127].
A closer look at the obtained Pt-specific chromatograms in the context of the size
calibration markers revealed that the retention times of the first 3 Pt peaks corresponded to
protein bound Pt-species, followed by 2 Pt peaks corresponding to hydrolysis products which
eluted near the inclusion volume. The last Pt peak eluted past the inclusion volume, and
corresponded to the parent drug. The first protein bound Pt peak (PP-1) co-eluted with Zn (data
not shown). This Zn peak likely corresponds to α2-macroglobulin, which is a Zn metalloprotein
40
that is known to interact with CP hydrolysis products [155]. Thus, PP-1 was assigned to a Pt-α2macroglobulin complex.
670 k Da 158 k Da 44 k Da 17 k Da 1.35 k Da
H-17-1
5 min
80
CP
60
40
Pt Intensity (CPS)
20
Pt-containing
hydrolysis
products
0
40
H-17-1
2 hr
CP
6
30
Protein bound
Pt-species
20
3
2
10
V0 1
Pt-containing
hydrolysis
products
5
4
0
400
600
800 1000 1200 1400 1600 1800 2000
Retention Time (s)
Figure 3.4: Representative Pt-specific chromatograms obtained for the analysis of H-17-1 spiked
with CP (0.04 mg/mL). The mixture was incubated at 37 °C and analyzed after 5 min and 2 hr.
Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13 µm particle size) at 22°C.
Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. Detector:
ICP-AES at 214.423 nm (Pt). Peaks 1-3: protein bound Pt-species, peaks 4-5: Pt-containing
hydrolysis products, peak 6: CP. The retention times of the molecular weight markers are
depicted on top.
41
Table 3.2: Areas of all Pt peaks expressed as % values of total Pt obtained after SEC-ICP-AES
analysis of human plasma spiked with CP after incubation at 37°C. * Corresponds to the average
of two determinations.
ID #
HSA
(g/L)
H-02
H-04
H-05
H-06
H-07
H-08
H-11
H-12
H-13
H-14
H-15
H-16
H-17
H-19
Avg ±
STD
43
46
35
40
41
40
44
44
41
50
39
43
40
44
42 ± 4
5 min
Protein
Bound
Pt
0
0
0*
0*
0*
0*
0
0*
0*
0*
0*
N/A
0
0
0±0
PtHydrolysis
Products
2.8
2.4
2.6*
2.1*
2.7*
2.0*
2.3
2.5*
2.5*
2.4*
2.4*
N/A
1.9
2.6
2.4 ± 0.3
Free
Drug
97.2
97.6
97.4*
97.9*
97.3*
98.0*
97.7
97.5*
97.5*
97.6*
97.6*
N/A
98.1
97.4
97.6 ±
0.3
2 hr
Protein
Bound
Pt
53.7
53.5
49.1*
55.3*
52.2
55.0*
53.5
52.3*
47.6*
49.4
53.1
54.7
53.7*
58.7
53.0 ±
2.8
PtHydrolysis
Products
17.1
16.2
17.9*
15.5*
17.1
14.6*
15.6
16.1*
16.9*
18.2
15.2
15.8
14.9*
11.4
15.9 ± 1.7
Free
Drug
29.2
30.3
33.0*
29.3*
30.6
30.5*
30.9
31.6*
35.5*
32.4
31.7
29.5
31.3*
29.9
31.1 ±
1.7
Since PP-3 co-eluted with HSA (this has also been previously observed) [127], it was
assigned to a Pt-HSA complex [67]. Due to the fact that CP derived hydrolysis products are
known to bind to Tf (see section 1.4.2) and Tf has a molecular mass of 79 kDa [150] which is
close to that of HSA (66.3 kDa), the inherently limited resolution offered by the employed SEC
column is incapable of delineating whether the Pt peak corresponds to Pt-Tf or Pt-HSA.
However, the major contribution of this Pt peak is attributed to Pt-HSA due to the higher affinity
of CP hydrolysis products for HSA over Tf
[60], as well as the > 10 fold higher molar
concentration of HSA in plasma compared to Tf (Table 2.1). PP-2 was assigned to Pt-HSA
multimer complexes because a previous study has demonstrated that the incubation of CP with
42
Table 3.3: A more detailed analysis of the Pt peak areas and retention times observed at the 2 hr
time point expressed as % values of total Pt obtained after SEC-ICP-AES analysis of human
plasma spiked with CP. * Corresponds to one analysis. **Corresponds to the average of two
analyses. The average difference between the analyses for PP-3 was 0.7% PP = Protein bound
Pt-species, HP = Pt-containing hydrolysis product.
ID #
PP-1
(tr=527 ±
9s)
PP-2
(tr=751 ±
8s)
H-02*
H-04*
H-05**
H-06**
H-07*
H-08**
H-11*
H-12**
H-13**
H-14*
H-15*
H-16*
H-17**
H-19*
Avg ±
STD
8.1
7.7
6.8
12.2
9.2
8.7
8.3
7.3
9.3
6.7
8.5
9.1
6.9
7.9
8.3 ± 1.4
13.5
11.7
11.2
12.6
13.1
14.8
11.9
12.5
9.7
11.6
12.6
12.4
13.3
13.2
12.4 ± 1.2
PP-3
HSA
(tr=890
± 7s)
32.1
34.2
31.1
30.5
29.9
31.5
33.2
32.5
28.6
31.0
32.0
33.1
33.6
37.6
32.2 ±
2.2
HP-1
(tr=1273 ±
20s)
HP-2
(tr=1388 ±
10s)
CP
(tr=1830
± 12s)
3.7
3.8
4.0
2.7
4.3
2.9
3.3
2.9
2.5
3.0
1.6
3.1
2.6
1.3
3.0 ± 0.8
13.3
12.5
13.9
12.8
12.8
11.7
12.3
13.1
14.4
15.2
13.6
12.7
12.3
10.0
12.9 ± 1.2
29.2
30.3
33.0
29.3
30.6
30.5
30.9
31.6
35.5
32.4
31.7
29.5
31.3
29.9
31.1 ±
1.7
HSA results in their formation [67]. Pt-peaks 4 & 5 likely correspond to Pt-containing hydrolysis
products (HP-1 and HP-2) because they eluted near the inclusion volume. HP-2, being the more
abundant species, was tentatively identified in a previous study as [PtClOH2(NH3)2]+ [127], since
the formation of the first hydrolysis product of CP involves the substitution of one chlorine atom
of CP by a H2O molecule (Figure 1.3). Any attempts to characterize this metabolite by ESI-MS
and APCI-MS in this previous study were unsuccessful, possibly due to low concentrations of
the Pt species as well as the high salt content in the collected fraction [127]. HP-2 was also the
only Pt peak other than free CP to be present at the 5 min time point (Figure 3.4). Based on the
43
smaller intensity of HP-1, the latter likely represents [Pt(OH2)2(NH3)2]+. The last Pt peak that
was detected eluted past the inclusion volume and was previously assigned to the parent drug
CP. This unusually long retention time is attributed to an unknown interaction between CP and
the stationary phase, and has also been observed on closely related stationary phases, such as
Superdex 75 [197].
In the context of relating the plasma [HSA] to the metabolism of CP (% Pt-bound HSA)
in all investigated plasma samples (n=14), no correlation was observed at the 2 hr time point
(Figure 3.5).
40
% Pt-bound HSA
38
36
R2 = 0.08
34
32
30
28
34
36
38
40
42
44
46
48
50
52
HSA (g/L)
Figure 3.5: HSA concentrations plotted against their corresponding Pt peak areas of Pt-bound
HSA expressed as a percentage.
3.3 Metabolism of CP in plasma of pediatric cancer patients
Blood was collected from 11 pediatric cancer patients and assigned I.D. #s (privacy
protection). All plasma samples were analyzed for creatinine, B.U.N., Tf, and HSA (Table 3.4).
44
Table 3.4: Concentration of HSA, Tf, creatinine, and B.U.N. in blood plasma of pediatric cancer
patients compared to healthy levels. The ranges pertaining to healthy children are depicted at the
bottom of the table.
ID #
C-01
C-02
C-03
C-04
C-05
C-06
C-07
C-08
C-09
C-10
C-11
Reference
range
Reference
range
Reference
range
Reference
range
Gender /
Age
Male / 13
Male / 3
Male / 15
Female / 15
Male / 14
Female / 12
Female / 14
Female / 16
Female / 14
Male / 18
Female / 3
M/F 0-5
HSA
(g/L)
28
34
30
21
26
28
30
28
28
29
8
33-48
M/F 6-12
33-48
M/F 13-14
33-48
M 15-18
F 15-18
33-48
Tf
(g/L)
1.48
2.19
1.70
0.93
1.23
1.97
1.73
0.81
N/A
2.05
0.26
2.003.60
2.003.60
2.003.60
2.003.60
Creatinine
(µmol/L)
24
23
63
30
44
28
37
44
34
65
18
20-60
B.U.N.
(mmol/L)
2.4
1.7
5.7
2.3
1.9
3.9
5.6
1.0
4.5
5.7
8.8
2.0-7.0
30-70
2.0-7.0
40-85
2.0-7.0
50-120
35-100
3.0-7.5
2.0-7.0
B.U.N. :
Creatinine
24.8
18.3
22.4
19.0
10.7
34.5
37.5
5.6
32.8
21.7
121.1
> 20 =
Dehydration
> 20 =
Dehydration
> 20 =
Dehydration
> 20 =
Dehydration
The concentrations for the measured plasma constituents were all below the reference
range or on a lower tier. These findings are in accord with earlier studies showing patients with
lung cancer becoming malnourished in part because of a lack of appetite [198].
3.3.1 Quality control of analytical results
The same exclusion criteria that were previously defined were applied to the results
obtained for plasma from pediatric cancer patients spiked with CP (see Appendix I). No results
obtained for the analysis of plasma were excluded from the discussion.
45
3.3.2 Cu-specific chromatograms
The retention times of ceruloplasmin were highly reproducible for all 11 cancer patient
plasma samples at both time points (Figure 3.6). Therefore, based on these results, all
simultaneously obtained Pt-specific chromatograms must be reliable.
670 k Da
5 min
n = 11
Ceruloplasmin
158 k Da 44 k Da 17 k Da 1.35 k Da
670 k Da
158 k Da 44 k Da 17 k Da 1.35 k Da
1000
Ceruloplasmin
2 hr
n = 10
Cu Intensity (CPS)
800
600
400
V0
V0
200
0
400
600
800
1000
1200
1400 400
Retention Time (s)
600
800
1000
1200
1400
Retention Time (s)
Figure 3.6: Representative SEC-ICP-AES derived Cu-specific chromatograms for the analysis
of 11 cancer patients spiked with CP (0.04 mg/mL). The mixture was incubated at 37 °C and
analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm
i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 324.754 nm (Cu). The retention times of the
molecular weight markers are depicted on top.
3.3.3 Fe and Zn-specific chromatograms
Since the Fe and Zn specific chromatograms that were obtained after the addition of CP
to plasma were very similar between the 5 min and the 2 hr time point, they are presented in
Appendix G & H. These data were accumulated since they can be simultaneously obtained and
46
because these results may help to explain some of the results obtained from the Ptchromatograms.
3.3.4 Pt-specific chromatograms
The Pt distribution in plasma that was determined for all 11 cancer plasma samples at the
5 min and the 2 hr time points are summarized in Table 3.5 and will be discussed first. A sample
Pt-chromatogram is shown in Figure 3.7 for C-11. A more detailed view of all detected Pt peaks
at the 2 hr time point is displayed in Table 3.6.
Table 3.5: Peak areas of all Pt peaks expressed as % values of total Pt obtained after SEC-ICPAES analysis of human plasma spiked with CP after incubation at 37°C.
HSA (g/L)
ID #
C-01
C-02
C-03
C-04
C-05
C-06
C-07
C-08
C-09
C-10
C-11
Avg ±
STD
28
34
30
21
26
28
30
28
28
29
8
26 ± 7
5 min
Protein
Bound
Pt
0
0
0
0
0
0
0
0
0
0
0
0±0
PtHydrolysis
Products
1.8
2.4
3.0
2.6
2.0
1.7
2.5
2.2
2.7
2.9
2.5
2.4 ± 0.4
Free
Drug
98.2
97.6
97.0
97.4
98.0
98.3
97.5
97.8
97.3
97.1
97.5
97.6 ±
0.4
2 hr
Protein
Bound
Pt
N/A
39.5
44.0
40.3
55.3
56.8
47.4
39.5
35.4
49.2
25.1
43.2 ±
9.5
PtHydrolysis
Products
N/A
23.1
19.2
24.1
14.1
14.1
19.2
19.4
26.9
19.2
32.6
21.2 ± 5.7
Free
Drug
N/A
37.4
36.8
35.6
30.6
29.1
33.3
41.1
37.8
31.6
42.3
35.6 ±
4.4
5 min after the addition of CP, no protein bound Pt was detected, 2.4 ± 0.4% eluted in the
form of hydrolysis products, and 97.6% remained as the free drug. At the 2 hr time point, 43.2 ±
47
670 k Da 158 k Da 44 k Da 17 k Da1.35 k Da
160
5 min
CP
140
120
100
80
60
Pt Intensity (CPS)
40
Pt-containing
hydrolysis
products
20
0
80
2 hr
CP
60
Pt-containing
hydrolysis
products
40
Protein bound
Pt-species
20
V0
0
400
600
800 1000 1200 1400 1600 1800 2000
Retention Time (s)
Figure 3.7: Representative Pt-specific chromatograms obtained for the analysis of C-11 spiked
with CP (0.04 mg/mL). The mixture was incubated at 37 °C and analyzed after 5 min and 2 hr.
Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13 µm particle size) at 22°C.
Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection volume: 500 µL. Detector:
ICP-AES at 214.423 nm (Pt). The retention times of the molecular weight markers are depicted
on top.
48
9.5% of the Pt was protein bound, with the majority bound to HSA (22.3 ± 5.7%), while 21.2 ±
5.7% eluted as hydrolysis products, and 35.6 ± 4.4% as the free drug.
Table 3.6: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of human plasma spiked with CP after incubation at 37°C for 2 hr. PP = Protein bound
Pt-species, PH = Pt-containing hydrolysis product.
ID #
C-01
C-02
C-03
C-04
C-05
C-06
C-07
C-08
C-09
C-10
C-11
Avg ±
STD
PP-1 (tr=
510 ± 9s)
N/A
6.4
10.1
9.7
7.2
13.0
11.2
8.4
11.7
13.6
10.8
10.2 ± 2.4
PP-2 (tr=
724 ± 8s)
N/A
8.9
9.8
8.8
21.3
15.5
11.2
7.9
7.0
11.6
5.5
10.8 ± 4.6
PP-3 HSA (tr=
868 ± 12s)
N/A
24.2
24.1
21.8
26.8
28.3
25.0
23.2
16.6
24.0
8.9
22.3 ± 5.7
PH-1 (tr=
1259 ± 15s)
N/A
4.7
3.3
5.6
2.3
2.4
3.8
3.1
6.3
3.1
7.2
4.2 ± 1.7
PH-2 (tr=
1377 ± 8s)
N/A
18.4
15.9
18.5
11.8
11.7
15.4
16.3
20.6
16.1
25.4
17.0 ± 4.1
CP (tr=
1820 ± 8s)
N/A
37.4
36.8
35.6
30.6
29.1
33.3
41.1
37.8
31.6
42.3
35.6 ± 4.4
3.4 Comparison of plasma from healthy adults with pediatric cancer plasma
The comparison of the Pt results for healthy adults (n=14) with pediatric cancer patients
(n=11) is depicted in Table 3.7. The results that were obtained for the 5 min time point are
virtually identical between the healthy adults and the pediatric cancer patients. In order to discuss
the differences at the 2 hr time point, it is most instructive to put it in a biochemical context.
Therefore, the results pertaining to CP will be discussed first, and then the hydrolysis products,
and lastly the protein bound Pt. In addition, it will be attempted to rationalize the obtained results
based on the molar ratio between CP and HSA, which had a range of 0.177-0.252 for the healthy
adults and 0.260-1.105 for the pediatric cancer patients.
49
Table 3.7: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from healthy adults (Avg. 42 g HSA/L) and pediatric cancer patients (Avg. 26
g HSA/L) spiked with CP after incubation at 37°C for 2 hr. PP = Protein bound Pt-species, HP =
Pt-containing hydrolysis product, H = Healthy adults, C = Pediatric cancer patients.
Time
PP-1
PP-2
HP-1
0±0
PP-3 Total
(HSA) PP
0±0
0±0
H - 5 min
n =13
C - 5 min
n = 11
H – 2 hr
n =14
C – 2 hr
n = 10
0±0
0±0
0±0
0±0
0±0
0±0
8.3 ±
1.4
10.2 ±
2.4
12.4 ±
1.2
10.8 ±
4.6
32.2 ±
2.2
22.3 ±
5.7
53.0 ±
2.8
43.2 ±
9.5
3.0 ±
0.8
4.2 ±
1.7
0±0
HP-2
Total
HP
2.4 ± 2.4 ±
0.3
0.3
2.4 ± 2.4 ±
0.4
0.4
12.9 ± 15.9 ±
1.2
1.7
17.0 ± 21.2 ±
4.1
5.7
CP
97.6 ±
0.3
97.6 ±
0.5
31.1 ±
1.7
35.6 ±
4.4
Overall, on average, 4.5% more of the parent CP as well as 5.3% more of the total HP
were present in the pediatric cancer patients compared to the healthy adults. Related to this, the
total protein bound Pt was 9.8% less in the pediatric cancer patients compared to the healthy
adults. In order to rationalize these findings, and taking into account the differences in molar
ratio between CP and HSA, it appears that a high molar ratio is associated with elevated
concentrations of hydrolysis products and a concomitant decrease in plasma protein bound Pt.
With regard to the individual hydrolysis products, the difference in HP-1 and HP-2 was 1.2%
and 4.1% higher in the pediatric cancer patients compared to the healthy adults. This corresponds
to a 32% increase in the total HP Pt area, and implies that the number of occupied binding sites
for HP is much lower in plasma compared to the maximum number of binding sites deducted
from experiments conducted with pure HSA and CP (~20 molecules of CP / HSA, Table 1.2).
With regard to protein bound Pt, the percentage of Pt bound to PP-1 was 1.9% higher in the
pediatric cancer patients, which is in contrast to the lower total protein bound Pt in this group.
This observation can be rationalized by the apparently increased concentration of α250
macroglobulin in the plasma from pediatric cancer patients compared to that in healthy controls
(see Appendix H). Based on the assumption that PP-2 and PP-3 both contain HSA bound Pt, it is
unsurprising that both Pt peak areas were lower in the pediatric cancer patients (1.6% and 9.9%)
compared to the healthy adults. In general, these results can be rationalized by a model outlined
in Figure 3.8.
Figure 3.8: Proposed model to link the observed in vitro results with (A) high HSA
concentration (bold font) in healthy controls and (B) lower concentrations of HSA in pediatric
cancer patients (normal font) with the increase in severe toxic side effects.
3.5 Comparison of pediatric cancer patient plasma with HSA fortified plasma
In order to corroborate the differences that were observed between the metabolism of CP
in healthy adults and pediatric cancer patients, the HSA concentration of plasma from individual
pediatric cancer patients (26 ± 7 g HSA /L HSA) was fortified with highly pure HSA to achieve
final plasma concentrations of 36 g HSA /L and 42 g HSA /L. At the 5 min time point, the Pt
distribution was largely independent of the HSA concentration in plasma. In order to discuss the
differences between the plasma from pediatric cancer patients and their HSA fortified plasma at
the 2 hr time point in a biochemical context, we will discuss the results pertaining to CP first,
51
then the HP, and lastly the protein bound Pt. It will be attempted to rationalize the obtained
results based on the molar ratio between CP and HSA, which had a range of 0.260-1.105 for the
unfortified pediatric cancer patient plasma, 0.246 for 36 g HSA /L, and 0.210 for 42 g HSA/L.
The Pt eluting as CP appeared to be independent of the HSA concentration in plasma
(Table 3.8). However, the Pt area corresponding to the total HP gradually decreased by 5.6%
upon fortification to 36 g HSA/L, and decreased by another 2.7% with 42 g HSA/L. In fact, the
Pt area corresponding to total HP in the HSA fortified plasma from pediatric cancer patients (42
g HSA/L) at the 2 hr time point was 12.9%, which was reasonably close to the value obtained for
the healthy adults (42 ± 4 g HSA/L) of 15.9%. Related to this, the total protein bound Pt
increased by 5.3% and another 4.1% with HSA plasma concentrations of 36 g HSA/L and 42 g
HSA/L, respectively. These results can be accounted for by the differences in the molar ratio
between CP and HSA, and the associated increased protein binding of the hydrolysis products.
Table 3.8: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from pediatric cancer patients (Avg. 26 g HSA/L) and the HSA fortified
plasma spiked with CP after incubation at 37°C for 2 hr. PP = Protein bound Pt-species, HP = Ptcontaining hydrolysis product, H = Healthy adults, C = Pediatric cancer patients.
Time / [HSA]
PP-1
PP-2
Total
PP
0±0
HP-1
0±0
PP-3
(HSA)
0±0
C - 5 min (Avg 26
g HSA/L) n =11
C - 5 min (36 g
HSA/L) n = 3
C – 5 min
(42 g HSA/L)
n=9
C – 2 hr (Avg 26 g
HSA/L) n =10
C – 2 hr (36 g
HSA/L) n = 10
C – 2 hr
(42 g HSA/L)
n = 10
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
10.2 ± 10.8 ± 22.3
2.4
4.6
5.7
9.9 ± 9.1 ± 29.6
3.0
3.3
2.5
10.4 ± 9.5 ± 32.8
2.6
2.8
1.6
0±0
HP-2
Total
HP
2.4 ± 2.4 ±
0.4
0.4
2.3 ± 2.3 ±
0.4
0.4
2.1 ± 2.1 ±
0.4
0.4
CP
97.6 ±
0.4
97.7 ±
0.4
98.0 ±
0.4
± 43.2 ± 4.2 ± 17.0 ± 21.2 ± 35.6 ±
9.5
1.7
4.1
5.7
4.4
± 48.5 ± 2.4 ± 13.2 ± 15.6 ± 35.9 ±
5.7
0.9
2.3
3.0
4.1
± 52.6 ± 1.8 ± 11.1 ± 12.9 ± 34.5 ±
4.3
0.6
2.1
2.5
3.0
52
With regard to the individual hydrolysis products, the Pt peak areas corresponding to HP1 and HP-2 gradually decreased by 2.4% and 5.9% at 42 g HSA/L respectively. These findings
further imply that the introduction of additional HSA binding sites for the HP in pediatric cancer
patient plasma may be used to decrease the fraction of free hydrolysis products in plasma. With
regard to protein bound Pt, the percentage of Pt bound to PP-1 was essentially unaffected by the
HSA concentration, which may be attributed to the constant α2-macroglobulin concentration in
the corresponding HSA spiked plasma samples. The amount of Pt that eluted as PP-2 first
decreased by 1.7% upon addition of HSA to 36 g HSA/L and remained essentially unchanged
upon further addition of HSA to 42 g HSA/L. This general trend is significantly different from
PP-3, which gradually increased by 10.5% over the same HSA range.
In the context of relating the [HSA] in plasma to results obtained for the metabolism of
CP over a 2 hr period, the concentration of HSA in the plasma from pediatric cancer patients was
correlated to the % Pt-bound HSA (Figure 3.9) with an R2 value of 0.79.
40
R2 = 0.79
35
% Pt-bound HSA
30
25
20
15
10
5
10
20
30
40
HSA (g/L)
Figure 3.9: HSA concentrations of pediatric cancer patients as well as their plasma fortified with
HSA (36 g HSA/L and 42 g HSA/L) plotted against their corresponding Pt peak areas of Ptbound HSA expressed as a %.
53
Despite the comparatively high standard deviations for the various Pt-bound protein
species (Table 3.8), the percent variation of PP-1 and PP-2 was low for individual patients. To
illustrate this, Table 3.9 and Table 3.10 provide all data that were obtained for patients C-11
(lowest HSA concentration – 8 g HSA/L) and C-02 (randomly chosen). For example, at the 2 hr
time point for C-11, PP-1 corresponded to 11.3 ± 1.2% of total Pt, and 6.4 ± 0.1% for C-02,
which is much lower than the standard deviation obtained for all samples (e.g., PP-1 is 3.0% at
36 g HSA/L). This trend was also evident with PP-2, as well as free CP.
Table 3.9: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from C-11 after being spiked with CP and varying amounts of HSA. PP =
Protein bound Pt-species, HP = Pt-containing hydrolysis product.
Time / [HSA]
PP-1
PP-2
Total
PP
0
HP1
0
HP2
2.5
Total
HP
2.5
CP
0
PP-3
HSA
0
5 min – No additional
HSA (8 g HSA/L)
5 min – 42 g HSA/L
2 hr – No additional
HSA (8 g HSA/L)
2 hr – 36 g HSA/L
2 hr – 42 g HSA/L
0
0
10.8
0
5.5
0
8.9
0
25.1
0
7.2
2.1
25.4
2.1
32.6
97.9
42.3
10.5
12.6
5.5
5.8
29.7
31.1
45.7
49.5
2.6
1.8
11.4
9.3
14.0
11.1
40.3
39.4
97.5
Table 3.10: Peak areas of all Pt peaks expressed as % of total Pt obtained after SEC-ICP-AES
analysis of plasma from C-02 after being spiked with CP and varying amounts of HSA. PP =
Protein bound Pt-species, HP = Pt-containing hydrolysis product.
Time / [HSA]
PP-1
PP-2
Total
PP
0
HP1
0
HP2
2.4
Total
HP
2.4
CP
0
PP-3
HSA
0
5 min – No additional
HSA (34 g HSA/L)
5 min – 42 g HSA/L
2 hr – No additional
HSA (34 g HSA/L)
2 hr – 36 g HSA/L
2 hr – 42 g HSA/L
0
0
6.4
0
8.9
0
24.2
0
39.5
0
4.7
2.7
18.4
2.7
23.1
97.3
37.4
6.2
6.4
7.6
7.9
27.6
30.6
41.4
45.0
3.6
2.5
17.2
15.6
20.8
18.0
37.7
37.0
54
97.6
Linear regression analysis (g HSA/L versus % Pt-bound HSA) was performed on all 11
pediatric cancer patients except for C-01 and C-03 because either insufficient amounts of plasma
were obtained or the plasma extinguished during analysis. The data are shown in Table 3.11. C05, C-06, and C-09 exhibited much different results. The underlying differences pertaining to
these particular samples are currently unknown.
Table 3.11: Linear regression analysis showing the increase of Pt-bound HSA per gram of HSA
added to plasma.
Sample
C-02
C-04
C-05
C-06
C-07
C-08
C-09
C-10
C-11
Average ± Std. Dev.
% Increase of Pt-bound HSA / g
of HSA added
0.73
0.69
0.37
0.27
0.59
0.62
1.30
0.72
0.68
0.68 ± 0.29
55
CHAPTER 4 PLASMA PROTEIN BINDING OF POTENTIALLY NOVEL METALBASED ANTICANCER COMPOUNDS
In order to potentially replace some of the established Pt-based anticancer drugs with
ones that exhibit less toxic side effects and are as effective, potential drug candidates must fulfill
desirable physical chemical properties. Among some of these, such as water solubility, an ideal
drug should rather selectively target cancer cells and be up-taken by them, while exerting less
severe toxic side effects than CP for example. Since virtually all anticancer drugs are
intravenously administered, the interaction of potential anticancer drug candidates with plasma
proteins is another extremely important criterion which determines a drug’s efficacy and safety
[45, 199]. We obtained metal-based compounds which exhibited high cytotoxicity (low IC50
values) from a collaborator and employed SEC-ICP-AES to conduct preliminary experiments to
assess the interaction of these Pt and Cu-based drugs with rabbit plasma in vitro, over a 1 hr
period.
4.1 Investigation of 56MESS and 56MESSCu
The C, Fe, and Pt-specific chromatograms that were obtained at the 5min and the 1 hr
time point for 56MESS revealed a single Pt peak which eluted past the inclusion volume (Figure
4.1). This behavior indicates that this Pt compound – which displayed a 10 times lower IC50 than
CP - either did not form adducts with plasma proteins within 1 hr or that the compound was
rapidly converted into a Pt species that did not interact with plasma proteins. This behavior is in
stark contrast to CP, which undergoes partial hydrolysis and plasma protein binding over the
same time period [79]. Given that 56MESS has been demonstrated to be able to intercalate into
the DNA in L1210 murine leukemia cells [181], our results indicate that in these cell culture
56
experiments, the transport of this compound across the cell membrane must involve a mechanism
that does not require plasma protein binding. Considering the hydrophobicity of the
phenanthroline-moiety of 56MESS, it is possible that its cell uptake involves passive diffusion
(Figure 1.11A). Interestingly, 56MESS has already been investigated in vivo and displayed no
antitumoral activity in BD-IX rats whatsoever [200]. These findings may be rationalized in terms
of the unselective uptake of 56MESS into all cells, therefore not exhibiting the desired selective
toxicity.
158 kDa 44 kDa 17 kDa
1.35 kDa
50
Carbon
Platinum
Iron
Transferrin
40
30
56MESS
15000
20
Ferritin
Vo
0
0
30000
C Intensity (CPS)
0
50
Carbon
Platinum
Iron
Albumin
40
150
Transferrin
30
56MESS
15000
20
Ferritin
Vo
100
50
10
5000
0
0
400
200
1 hr
20000
10000
50
10
5000
25000
100
Fe Intensity (CPS)
10000
Pt Intensity (CPS)
150
20000
Pt Intensity (CPS)
C Intensity (CPS)
25000
200
5 min
Albumin
Fe Intensity (CPS)
670 kDa
30000
600
800
1000
1200
1400
1600
0
1800
Retention Times (s)
Figure 4.1: Representative C, Fe, and Pt-specific chromatograms obtained for the analysis of
rabbit plasma spiked with 56MESS (100 µmol/L). The mixture was incubated at 37 °C and
samples were analyzed after 5 min and 1 hr . Stationary phase: Superdex 200 10/300 GL column
(30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0
mL/min. Injection volume: 500 µL. The retention times of the molecular markers are depicted on
top.
57
In contrast, the results that were obtained for the closely related 56MESSCu indicated a
completely different solution chemistry than 56MESS at both time points (Figure 4.2).
Compared to the control plasma (data not shown), the most intense Cu peak in the 56MESSCu
spiked rabbit plasma corresponded to a Cu-entity (either 56MESSCu or a Cu containing
degradation product bound thereof) to a plasma protein with a molecular weight of > 44kDa.
Since this molecular weight corresponds to the elution of Tf and HSA, 56MESSCu and/or its Cu
containing degradation product rapidly hydrolyzes in plasma and the generated Cu moieties then
bind/coordinate to highly abundant plasma proteins.
The strikingly different behavior of
56MESSCu compared to 56MESS is noteworthy and can be rationalized by the known lability of
Cu-NH2 bonds in the former compared to the analogous Pt bonds in the latter.
670 kDa
158 kDa 44 kDa 17 kDa
1.35 kDa
30000
1600
200
1200
150
C Intensity (CPS)
20000
15000
800
10000
400
Vo
100
50
Fe Intensity (CPS)
Carbon
Copper
Iron
25000
Cu Intensity (CPS)
5 min
5000
0
30000
0
0
1600
200
1200
150
15000
800
10000
5000
400
Vo
0
0
400
600
800
1000
1200
1400
1600
100
50
Fe Intensity (CPS)
C Intensity (CPS)
20000
Cu Intensity (CPS)
1 hr
Carbon
Copper
Iron
25000
0
1800
Retention Times (s)
Figure 4.2: Representative C, Fe, and Cu-specific chromatograms obtained for the analysis of
rabbit plasma spiked with 56MESSCu (100 µmol/L). The mixture was incubated at 37 °C and
samples were analyzed after 5 min and 1 hr . Stationary phase: Superdex 200 10/300 GL column
(30x1.0 cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0
mL/min. Injection volume: 500 µL. The retention times of the molecular markers are depicted on
top.
58
4.2 Investigation of DiPy and SOOS
The C, Fe, and Pt-specific chromatograms that were obtained at the 5 min and the 1 hr
time point for DiPy revealed a single Pt peak (Fig 4.3), the retention time of which indicated that
this Pt compound, unlike 56MESS, rapidly forms adducts with plasma proteins (within 5 min).
Given that DiPy and/or its hydrolysis products appear to form adducts with HSA and/or Tf (coelution with Fe), our results suggest that it may possibly enter cells via the Tf-mediated uptake
pathway.
158 kDa 44 kDa 17 kDa
1.35 kDa
5 min
Carbon
Platinum
Iron
200
40
150
20000
30
15000
20
10000
Vo
100
50
Fe Intensity (CPS)
C Intensity (CPS)
25000
50
Pt Intensity (CPS)
670 kDa
30000
10
5000
0
0
0
50
200
30000
1 hr
Carbon
Platinum
Iron
40
30
15000
20
10000
Vo
50
10
5000
0
0
400
100
Fe Intensity (CPS)
150
20000
Pt Intensity (CPS)
C Intensity (CPS)
25000
600
800
1000
1200
1400
1600
0
1800
Retention Times (s)
Figure 4.3: Representative C, Fe, and Pt-specific chromatograms obtained for the analysis of
rabbit plasma spiked with DiPy (100 µmol/L). The mixture was incubated at 37 °C and samples
were analyzed after 5 min and 1 hr . Stationary phase: Superdex 200 10/300 GL column (30x1.0
cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. The retention times of the molecular markers are depicted on top.
59
Unlike the other three compounds, SOOS displayed poor solubility in aqueous buffers.
Although it appeared that the compound had not dissolved, the solution was still mixed with the
rabbit plasma, and then injected onto the SEC column. After injection, the backpressure of the
column significantly increased due to a precipitate forming on the column, and the ICP plasma
extinguished. The 1 hr time point was missed while trying to stabilize the ICP plasma. After 4 hr,
another injection was performed and results were obtained (Figure 4.4). Since precipitate was
forming on the column, further injections with SOOS were not performed. Given that SOOS
and/or its hydrolysis products appear to form adducts with HSA and/or Tf (co-elution with Fe),
our results suggest that it may possibly enter cells via the Tf-mediated uptake pathway.
SOOS
670 kDa
158 kDa 44 kDa 17 kDa
1.35 kDa
35000
30
180
4 hr
30000
Carbon
Platinum
Iron
20000
15
15000
10
10000
5
120
100
80
60
Fe Intensity (CPS)
140
20
Pt Intensity (CPS)
25000
C Intensity (CPS)
160
25
40
5000
20
0
0
0
-5
400
600
800
1000
1200
1400
1600
1800
Retention Times (s)
Figure 4.4: Representative C, Fe, and Pt-specific chromatograms obtained for the analysis of
rabbit plasma spiked with SOOS. The mixture was incubated at 37 °C and samples were
analyzed after 4 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13 µm
particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection
volume: 500 µL. The retention times of the molecular markers are depicted on top.
60
CHAPTER 5 CONCLUSIONS
5.1 Effect of the plasma HSA concentration on the metabolism of CP in vitro
The analysis of CP spiked blood plasma from 14 healthy adults (control, 42 ± 4 g HSA/L)
and 11 pediatric cancer patients (26 ± 7 g HSA/L) at the 5 min and 2 hr time point revealed
differences in the Pt distribution and therefore the metabolism of CP. Since HSA is known to
play a key role in the metabolism of CP in plasma, the observed differences were rationalized in
terms of the different HSA concentrations between healthy adults and pediatric cancer patients.
In general and at the 2 hr time point, a decrease in the HSA concentration in plasma resulted in
less Pt being present in form of Pt-protein adducts (including HSA). Concomitantly, an increased
percentage of total Pt was present in form of free hydrolysis products.
To corroborate these results, HSA was added to plasma from individual cancer patients to
achieve final concentrations of 36 and 42 g HSA/L, and the metabolism of CP was determined as
outlined above. Overall, the metabolism of CP in the HSA fortified plasma was rather similar to
that of plasma in healthy adults. These experiments conclusively demonstrate that HSA plays an
important role in the metabolism of CP in human plasma in vitro. More importantly, our in vitro
results indicate that the concentration of biologically highly reactive free CP hydrolysis products
in human plasma may be decreased by the administration of HSA to pediatric cancer patients.
Since it is presently unknown which Pt species are predominantly responsible for exerting its
toxic side effects in patients and given that circumstantial evidence suggests that Pt-HAS adducts
are associated with less toxic side effects than CP [143], our in vitro experiments suggest that
these CP derived hydrolysis products may play a more critical role than is currently
acknowledged. Furthermore, our results represent an important first step towards optimizing the
61
treatment protocol of cancer patients that are administered with CP to mitigate the severe toxic
side effects.
62
5.2 Plasma protein binding of potentially novel metal-based anticancer compounds
Since virtually all anticancer drugs are intravenously administered, the interaction of
potential anticancer drugs with plasma proteins represents one important criterion (in addition to
IC50, Kd, etc.) which determines the drug’s efficacy and its toxic side effects. In order to assess
the comparative biochemical transformations of 4 metallo-compounds (Pt-containing
compounds: 56MESS, DiPy, and SOOS, and a Cu-containing compound: 56MESSCu) in
plasma, we added pharmacologically relevant doses of each of these metallo-compounds to
rabbit plasma, and analyzed it by SEC-ICP-AES after 5 min and 1 hr. 56MESS, or a Pt
containing degradation product thereof, did not form adducts with plasma proteins within 1 hr,
suggesting that the transport of this anticancer active metallo-compound across the cell
membrane may be driven by its hydrophobicity and does not involve plasma proteins.
Interestingly, the exchange of the Pt metal center in this compound for Cu (56MESSCu)
produced drastically different results in the sense that this metal based compound or a Cu
containing hydrolysis product thereof bound to HSA and/or Tf. This result can be rationalized in
terms of the known lability of Cu-NH2 bonds compared to the analogous Pt bonds which are
more stable. The interaction of 56MESSCu with plasma proteins and its established cytotoxicity
suggest that this metal based drug may enter cells via the Tf-mediated uptake pathway. The
addition of DiPy (5 min and 1 hr) and SOOS (4 hr) produced results that were very similar to
those of 56MESSCu.
63
CHAPTER 6 FUTURE WORK
SEC-ICP-AES represents a unique tool to probe the metabolism of metal-based drugs in
plasma in vitro and also potentially in vivo. In particular, one can simultaneously observe the
metabolism of the metal-based drug of choice, as well as potential perturbations caused by the
drug at the Cu, Fe, and Zn metalloprotein level which can possibly be linked to side effects. One
important next step would be to probe the interaction of CP with varying levels of HSA in vivo in
animal studies and/or patients. This would allow one to determine how quickly all Pt metabolites
disappear from the blood plasma. If this information were combined with the potential different
distributions of Pt in the organs of the animal, it could potentially provide insight into the toxic
side effects that are caused by CP. Thus, it may support the development of new treatment
protocols for optimizing the treatment of cancer patients with CP to mitigate the toxic side
effects. Another potential application would be to establish the molecular basis by which
ameliorating agents (e.g. STS, NAC, and D-methionine) mitigate some of the toxic side effects
of CP in vivo. To that end, previous studies have shown that STS will greatly reduce the amount
of CP based Pt-bound proteins as well as Pt-hydrolysis products by producing a Pt-STS complex
which is likely to play a role in the mitigation of these toxic side effects. By systematically
investigating the rate of the disappearance of the hydrolysis products in vivo after administration
with an ameliorating agent, one can investigate the effectiveness of these compounds. Since
information regarding the cytotoxicity of the Pt-ameliorating agent complexes is unknown, these
studies should be combined with the investigation of their cytotoxic properties on cell lines as
well. Obviously, the aforementioned also pertains to the study of novel metal-based drugs whose
metabolism can be investigated in vitro before more challenging and expensive in vivo
64
experiments are conducted. In addition, we can also investigate the fate of two metal-based drugs
that contain two different metals in vivo.
65
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APPENDIX A
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78
APPENDIX B
79
80
81
82
APPENDIX C
83
84
85
86
APPENDIX D
87
88
89
APPENDIX E
90
APPENDIX F
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93
APPENDIX G
670 kDa
158 kDa
44 kDa 17 kDa
670 kDa
1.35 kDa
100
5 min
n=14
Trans fe rrin
158 kDa
44 kDa 17 kDa
1.35 kDa
2 hr
n=14
Trans fe rrin
Fe Intensity (CPS)
80
60
40
Fe rritin
Fe rritin
20
V0
V0
0
400
600
800
1000
1200
1400 400
600
800
1000
1200
1400
Retention Time (s)
Retention Time (s)
SEC-ICP-AES derived Fe-specific chromatograms for the analysis of 14 plasma samples from
healthy controls spiked with CP (0.04 mg/mL). The mixtures were incubated at 37 °C and
analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm
i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 259.940 nm (Fe). The retention times of the
molecular markers are depicted on top of the figure.
94
670 k Da
5 min
n = 11
Transferrin
300
Fe Intensity (CPS)
670 k Da
158 k Da 44 k Da 17 k Da 1.35 k Da
350
158 k Da 44 k Da 17 k Da 1.35 k Da
2 hr
n = 10
Transferrin
250
200
Ferritin
150
Ferritin
100
V0
V0
50
0
400
600
800
1000
1200
1400 400
Retention Time (s)
600
800
1000
1200
1400
Retention Time (s)
SEC-ICP-AES derived Fe-specific chromatograms for the analysis of 11 pediatric cancer
patients spiked with CP (0.04 mg/mL). The mixtures were incubated at 37 °C and analyzed after
5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm i.d., 13 µm
particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min. Injection
volume: 500 µL. Detector: ICP-AES at 259.940 nm (Fe). The retention times of the molecular
markers are depicted on top of the figure.
95
APPENDIX H
670 kDa
44 kDa 17 kDa
158 kDa
670 kDa
1.35 kDa
60
1.35 kDa
2 hr
n=14
5 min
n=14
Album in
50
Zn Intensity (CPS)
44 kDa 17 kDa
158 kDa
Album in
40
2-m acroglobulin
2-m acroglobulin
Extrace llular
CuZnSOD
30
Extrace llular
CuZnSOD
20
V0
V0
10
0
400
600
800
1000
1200
1400 400
Retention Time (s)
600
800
1000
1200
1400
Retention Time (s)
SEC-ICP-AES derived Zn-specific chromatograms for the analysis of 14 plasma samples from
healthy controls spiked with CP (0.04 mg/mL). The mixtures were incubated at 37 °C and
analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0 cm
i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 213.856 nm (Zn). The retention times of the
molecular markers are depicted on top of the figure.
96
670 k Da
Zn Intensity (CPS)
50
670 k Da
158 k Da 44 k Da 17 k Da 1.35 k Da
60
2-m acroglobulin
5 min
n = 11
Album in
158 k Da 44 k Da 17 k Da 1.35 k Da
2 hr
n = 10
Album in
2-m acroglobulin
40
30
20
Extrace llular
CuZnSOD
Extrace llular
CuZnSOD
V0
V0
10
0
400
600
800
1000
1200
1400 400
Retention Time (s)
600
800
1000
1200
1400
Retention Time (s)
SEC-ICP-AES derived Zn-specific chromatograms for the analysis of 11 plasma samples from
pediatric cancer patients spiked with CP (0.04 mg/mL). The mixtures were incubated at 37 °C
and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0
cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 213.856 nm (Zn). The retention times of the
molecular markers are depicted on top of the figure.
.
97
APPENDIX I
670 kDa
158 kDa
44 kDa 17 kDa
1.35 kDa
50000
n = 11
HSA
C Intensity (CPS)
40000
30000
20000
10000
V0
0
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Retention Time (s)
SEC-ICP-AES derived C-specific chromatograms for the analysis of 11 plasma samples from
pediatric cancer patients spiked with CP (0.04 mg/mL). The mixtures were incubated at 37 °C
and analyzed after 5 min and 2 hr. Stationary phase: Superdex 200 10/300 GL column (30x1.0
cm i.d., 13 µm particle size) at 22°C. Mobile phase: PBS buffer (pH 7.4). Flow rate: 1.0 mL/min.
Injection volume: 500 µL. Detector: ICP-AES at 193.091 nm (C). The retention times of the
molecular markers are depicted on top of the figure.
98