UNIVERSITY OF GHENT
FACULTY OF PHARMACEUTICAL SCIENCES
Université de Lyon, France, Université Claude Bernard Lyon 1,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon
Département de Pharmacie Clinique, Pharmacocinétique et d’Evaluation du médicament.
VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD FOR THE DETERMINATION OF IMPDH ACTIVITY IN
PRELIMINARY STUDIES OF THE ENZYMATIC CONDITIONS
UNIVERSITY OF GHENT
FACULTY OF PHARMACEUTICAL SCIENCES
Université de Lyon, France, Université Claude Bernard Lyon 1,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon
Département de Pharmacie Clinique, Pharmacocinétique et d’Evaluation du médicament.
Professeur R. Boulieu
Academic year 2009 – 2010
VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD FOR THE DETERMINATION OF IMPDH ACTIVITY IN
ERYTHROCYTE LYSATE.
PRELIMINARY STUDIES OF THE ENZYMATIC CONDITIONS
Kirsten VANDERCRUYSSEN
First master in drug devoloppment
Promoter
Prof.Dr. S. Van Calenbergh
Commisaris
Dr. K. Boussery
Prof. T. De Beer
FACULTY OF PHARMACEUTICAL SCIENCES
Université de Lyon, France, Université Claude Bernard Lyon 1,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon
Département de Pharmacie Clinique, Pharmacocinétique et d’Evaluation du médicament.
VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD FOR THE DETERMINATION OF IMPDH ACTIVITY IN
PRELIMINARY STUDIES OF THE ENZYMATIC CONDITIONS
UNIVERSITY OF GHENT
FACULTY OF PHARMACEUTICAL SCIENCES
Université de Lyon, France, Université Claude Bernard Lyon 1,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon
Département de Pharmacie Clinique, Pharmacocinétique et d’Evaluation du médicament.
VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD FOR THE DETERMINATION OF IMPDH ACTIVITY IN
PRELIMINARY STUDIES OF THE ENZYMATIC CONDITIONS
UNIVERSITY OF GHENT
FACULTY OF PHARMACEUTICAL SCIENCES
Université de Lyon, France, Université Claude Bernard Lyon 1,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon
Département de Pharmacie Clinique, Pharmacocinétique et d’Evaluation du médicament.
Professeur R. Boulieu
Academic year 2009 – 2010
VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD FOR THE DETERMINATION OF IMPDH ACTIVITY IN
ERYTHROCYTE LYSATE.
PRELIMINARY STUDIES OF THE ENZYMATIC CONDITIONS
Kirsten VANDERCRUYSSEN
First master in drug devoloppment
Promoter
Prof.Dr. S. Van Calenbergh
Commisaris
Dr. K. Boussery
Prof. T. De Beer
FACULTY OF PHARMACEUTICAL SCIENCES
Université de Lyon, France, Université Claude Bernard Lyon 1,
Institut des Sciences Pharmaceutiques et Biologiques de Lyon
Département de Pharmacie Clinique, Pharmacocinétique et d’Evaluation du médicament.
VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD FOR THE DETERMINATION OF IMPDH ACTIVITY IN
PRELIMINARY STUDIES OF THE ENZYMATIC CONDITIONS
COPYRIGHT
“The author and the promoters give the authorization to consult and to copy parts of this thesis for
personal use only. Any other use is limited by the laws of copyright, especially concerning the
obligation to refer to the source whenever results from this theses are cited.”
May 18, 2010
Promotor Author
Prof.Dr. S. Van Calenbergh Kirsten Vandercruyssen
Acknowlodgments
Je souhaite remercier professeur Boulieu Rosalyne pour me donner l’occasion pour faire mon
stage du reserche à l’université Claude Bernard Lyon 1, ISPB. Je tiens aussi à remercier
l’assistent Jean Paul Salvi pour l’accompagnement pendant tout mon stage.
Un grand merci à Claudia, ma collègue Italienne qui était présente pendant mon stage, pour
la bonne ambiance et pour des beaux moments au laboratorium. Je veux aussi remercier des
autres étudiants et mes amis à Lyon, tout particulierement Laura, pour un expérience unique
d’érasmus à Lyon.
Merci à ma famille de me donner l’occasion pour y aller et de m’avoir supporter depuis le
début de mes études. Aussi un merci pour mes meilleurs amis belges de me supporter quand
c’était un peu difficile.
Finalement, je voudrais dire merci au Professeur Serge Van Calenbergh, directeur de ma
thèse, pour s’occuperer des corrections de ma thèse et pour me donner les bons conseils.
Kirsten Vandercruyssen
1 INTRODUCTION……………………………………………………………………….1
1.1 GENERAL INTRODUCTION……………………..…………………………………...1
1.2 MYCOPHENOLIC ACID………………………………................................................2
1.2.1 General introduction of mycophenolic acid…………...…………………….3
1.2.2 Mechanism…………………………………………………………………….3
1.2.2.1 Effects of MPA on proliferation of T-lymphocytes…………………..4
1.2.2.2 Effects of MPA on the production of antibodies……………………...5
1.2.3 Pharmacokinetics of MMF………………………………………………….6
1.2.3.1 Absorption…………………………………………………………….6
1.2.3.2 Distrubution……...……………………………………………………6
1.2.3.3 Metabolism……………………………………………………………6
1.2.3.4 Elimination……………………………………………………………7
1.2.3.5 Enterohepatic recirculation……………………………………………7
1.2.3.6 Drug interaction……………………………………………………….8
1.2.4 Adverse effects………………………………………………………………...8
1.3 INOSINE MONOPHOSPHATE DEHYDROGENASE………………………………..8
1.3.1 Characteristics………………………………………………………………...8
1.3.1.1 Mechanism of conversion of IMP to XMP………………………….10
1.3.2 Reaction of MPA with IMPDH……………………………………………..10
1.4 DETERMINATION OF THE ACTIVITY OF IMPDH……………………………….11
1.4.1 Radiolabeled assay…………………………………………………………..12
1.4.2 Chromatographic method…………………………………………………..12
1.4.2.1 Preparation of the samples…………………………………………...12
1.4.2.2 Enzymatic conditions………………………………………………..13
TABLE OF CONTENT
1.4.2.3 Chromatographic conditions…………………………………………13
1.4.3 Conclusion........................................................................................................16
2 GOAL……………………………………………..…………………………………..…18
3 MATERIALS AND METHODS………………………………………………………18
3.1 CHROMATOGRAPHIC SYSTEM……………………………………………………18
3.1.1 Stationary phase……………………………………………………………..18
3.1.2 Mobile phase…………………………………………………………...…….19
3.1.3 Detector………………………………………………………………………19
3.2 MATERIALS…………………………………………………………………………..20
3.3 VALIDATION OF THE METHOD…………………………………………………...21
3.3.1 Selectivity…………………………………………………………………….21
3.3.2 Linearity……………………………………………………………………...21
3.3.3 Accuracy and precision (repeatability and reproducibility)……………...22
3.3.4 Limit of quantification………………………………………………………23
3.4 INCUBATION CONDITIONS OF THE SAMPLES………………………………….24
3.4.1 Pretreatment of blood samples……………………………………………...24
3.4.2 Enzymatic conditions………………………………………………………..24
3.4.2.1 Incubation conditions………………………………………………..25
4 RESULTS……………………………………………………………………………….26
4.1 VALIDATION OF THE METHOD…………………………………………………...26
4.1.1 Selectivity…………………………………………………………………….26
4.1.2 Linearity……………………………………………………………………...27
4.1.2.1 Calibration curve of XMP…………………………………………...27
4.1.2.2 Calibration curve of IMP…………………………………………….28
4.1.3 Accuracy and precision (repeatability and reproducibility).......................30
4.2 ENZYMATIC CONDITIONS…………………………………………………………32
4.2.1 Influence of KCl………………………………………………………....….32
4.2.2 Influence of HClO4……………….…………………………………………33
4.2.3 Assays of previously reported publications………..………………………33
4.2.4 Influence of the pretreatment of the erythrocyte lysate…………………..34
4.2.5 Blank samples………………………………………………………………..35
5 DISCUSSION…………………………………………………………………………...36
5.1 VALIDATION OF THE METHOD…………………………………………………...36
5.2 ENZYMATIC CONDITION…………………………………………………………..37
6 CONCLUSION…………………………………………………………………………39
7 LIST OF REFERENCES………………………………………………………………40
LIST OF ABBREVIATIONS
6-TGN: 6-thioguanine nucleotide
AcMPAG: Acyl-MPA-glucuronide
ACN: Acetonitrile
AMP: Adenosine monophosphate
AUC: Area under the curve
CEDIA: Cloned Enzyme Donor Immunoassay
CMV: Cytomegalovirus
CV: Coefficient of Variation
dGTP: Deoxyguanosine triphosphate
DTT: DiThiothreitol
EDTA: Ethylenediaminetetraacetic acid
EMIT: Enzyme Multiplied Immunoassay Technique
ESI: Electrospray Ionisation
GMP : Guanosine 5‘-monophosphate
GTP : Guanosine 5’-triphosphate
HClO4 : Perchloric acid
HPLC: High Performance Liquid Chromatography
IMP : Inosine 5’-monophosphate
IMPDH : Inosine 5’monophosphate dehydrogenase
LC-MS/MS: Liquid Chromatography-tandem mass spectrometry
LoD: Limit of detection
LoQ: Limit of quantification
MMF : Mycophenolate mofetil
MPA : Mycophenolic acid
MPAG: 7-O-MPA-glucuronide
NAD+: Nicotinamide adenine dinucleotide
PE: Percentage of error
PMBC: Peripheral blood mononuclear cells
RP-HPLC: Reversed Phase-High Performance Liquid Chromatography
Rs : Separation coefficient
SD : Standard Deviation
TBAHS : Tetrabutylamoniumhydrogensulphate
TDM : Therapeutic Drug Monitoring
TEA: Triethylamine
UGT: Uridine diphosphate glucuronosyltransferase
XMP: Xanthosine 5’-monophosphate
1
1 INTRODUCTION
1.1 GENERAL INTRODUCTION
When an allograft organ transplantation is carried out, there exists a great risk of organ
rejection. Because the immune system of the receiver recognizes the heart, liver or kidney
transplant as foreign or non-self. Therefore, it is important to prevent acute and chronic
allograft rejection by giving prophylactic medication, i.e., immunosuppressive drugs. Several
classes of immunosuppressive agents exist, but most commonly used is a combination therapy
consisting of three different drugs, i.e., a corticosteroid, cyclosporine or azathioprine and
mycophenolic acid (MPA).
Corticosteroids, such as beclomethason, prednisone and methylprednisolone, have an
immunosuppressive and anti-inflammatory effect. They decrease the sensitivity of the tissues
receptors and also inhibit antigen presentation, cytokine production and proliferation of
lymphocytes, thereby inhibiting rejection.
Azathioprine is a purine-antagonist. First it becomes metabolized to 6-mercaptopurine,
followed by a second metabolic transformation to 6-thioguanine nucleotides, which are the
intracellular active metabolites, which are incorporate into nucleic acids, thereby causing
inhibition of the DNA synthesis and decreased immune cell proliferation.
Cyclosporine is a cyclic polypeptide consisting of 11 amino acids with a powerful
immunosuppressive effect. It interferes with the interleukine-2 (IL-2) gene transcription,
causing reversible inhibition of the activation and proliferation of cytotoxic T-cells. (Haglund
et al., 2007) (http://www.fk.cvz.nl)
Mycophenolic acid is the third component in the combination therapy. Its activity
involves the inhibition of inosine 5’-monophosphate dehydrogenase (IMPDH), a key-enzyme
in the de novo biosynthesis of the purines. Correct dosing of MPA is not trivial, since it has a
relatively narrow therapeutic window. Under- and overdosing of the drug may have serious
2
consequences including an increased risk of rejection and adverse reactions (Maiguma, 2009).
Moreover, MPA shows wide interpatient variability. Polymorphism exists in the gene
encoding for IMPDH, which leads to variability in its enzymatic activity. Therefore
therapeutic drug monitoring has been developed to establish correct dosing for each patient
individually. However, only determining the plasma concentration of MPA and its
metabolites is not enough. Assessing IMPDH-activity has become a new approach, which
should allow a more precise dosing of MPA. (Weimert et al., 2007)
Different methods have been investigated to determine the activity of IMPDH. The
goal of this work is to develop a chromatographic method. In a first part an introduction on
the drug MPA, the enzyme IMPDH and several analytical methods for IMPDH activity
determination are fully described. The second part deals with our attempts to develop an
analytical method for the determination of IMPDH activity, based on a RP-HPLC assay.
1.2 MYCOPHENOLIC ACID
1.2.1 General introduction of mycophenolic acid
Mycophenolic acid (MPA), discovered in 1893 as a fermentation product of Penicillium
brevicompactum, is an immunosuppressive drug. It was previously designed as an anti-cancer
agent, but is now generally used to prevent organ rejection after a kidney, liver or heart
transplantation. (Allison et al., 2005)
Because of its low bioavailability, a prodrug of MPA has been developed:
mycophenolate mofetil (MMF), also named Cellcept ®. (FIGURE 1.1) It is a 1,4-
morpholinoethyl ester of mycophenolic acid (MPA). MMF is used in tritherapy with
cyclosporine or tacrolimus, plus corticosteroids to significantly reduce acute rejection of the
transplanted organ. There are several formulations available for oral administration, such as
capsules, tablets and a powder for suspension. An intravenous formulation can also be
applied, when the patient is unable to take oral medication. (Armstrong et al., 2005)
R = : Mycophenolate mofetil (MMF)
FIGURE 1
Another type of prodrug is the enteric
sodium salt of MPA. It was designed for delayed release
al. 2009)
1.2.2 Mechanism
MPA is a potent, selective inhibitor of inosine monophosphate dehydrogenase
(IMPDH). The binding of MPA with the enzyme is reversible and non
catalyses the rate-limiting step
(FIGURE 1.2.) Inhibition leads to a depletion in the guanosine nucleo
indispensable for the synthesis of DNA and RNA. As a result, the proliferation of T and B
lymphocytes is inhibited because these cells
like other cells do, and thus
inhibition of the proliferation of B
immunoglobulins. (Allison et al., 2005)
Another consequence of depletion of the guanosine nucleotides, is that glycosylation
of adhesion molecules on lymphocytes and mono
molecules are involved in intracellular adhesion to endothelial ce
reduces recruitment of leukocytes to sites of infla
al.,2005) 3
R = H: Mycophenolic acid (MPA)
R = : Mycophenolate mofetil (MMF)
FIGURE 1.1.: STRUCTURE OF MPA AND MMF
Another type of prodrug is the enteric-coated mycophenolate sodium or Myfortic ®, a
sodium salt of MPA. It was designed for delayed release of the active substance.
MPA is a potent, selective inhibitor of inosine monophosphate dehydrogenase
(IMPDH). The binding of MPA with the enzyme is reversible and non-competitive. IMPDH
limiting step in the de novo biosynthesis of purine nucleotides.
Inhibition leads to a depletion in the guanosine nucleotides pools, which are
the synthesis of DNA and RNA. As a result, the proliferation of T and B
because these cells do not possess a salvage pathway for guan
like other cells do, and thus depend on IMPDH-activity for cell division
iferation of B-lymphocytes leads to a reduced production of
llison et al., 2005)
Another consequence of depletion of the guanosine nucleotides, is that glycosylation
of adhesion molecules on lymphocytes and monocyte glycoproteins is inhibited.
molecules are involved in intracellular adhesion to endothelial cells. By this action, MPA
reduces recruitment of leukocytes to sites of inflammation and graft rejection.
R = : Mycophenolate mofetil (MMF)
coated mycophenolate sodium or Myfortic ®, a
of the active substance. (Glander et
MPA is a potent, selective inhibitor of inosine monophosphate dehydrogenase
competitive. IMPDH
osynthesis of purine nucleotides.
tides pools, which are
the synthesis of DNA and RNA. As a result, the proliferation of T and B
do not possess a salvage pathway for guanosine,
for cell division. In addition,
a reduced production of
Another consequence of depletion of the guanosine nucleotides, is that glycosylation
cyte glycoproteins is inhibited. These
lls. By this action, MPA
mmation and graft rejection. (Allison et
FIGURE 1.2.: DE NOVO PATHWAY
CENTRAL POSITION OF IONISINE MONOPHOSPH
ACID INHIBITS IMPDH, THEREBY DEPLETING GUANOSINE MONOPHOPHATE
(GMP), GUANOSINE TRIPHOSPHATE (GTP) AND DEOXYGUANOSINE
TRIPHOSPHATE (dGTP) (C. Allison and M.Egui, 2005)
1.2.2.1 Effects of MPA on proliferation of T
MPA uses two distinct mechanisms to reduce the proliferation of T
First, T-lymphocytes do not have a salvage pathway,
novo guanosine nucleotide synthesis. Second, these cells have a different ex
isoform of IMPDH. (Allison et al., 2005)
4
DE NOVO PATHWAY FOR PURINE BIOSYNTHESIS, SHOWING
POSITION OF IONISINE MONOPHOSPHATE (IMP). MY
, THEREBY DEPLETING GUANOSINE MONOPHOPHATE
(GMP), GUANOSINE TRIPHOSPHATE (GTP) AND DEOXYGUANOSINE
(C. Allison and M.Egui, 2005)
Effects of MPA on proliferation of T-lymphocytes
distinct mechanisms to reduce the proliferation of T
lymphocytes do not have a salvage pathway, and thus completely dependent o
novo guanosine nucleotide synthesis. Second, these cells have a different ex
(Allison et al., 2005)
YNTHESIS, SHOWING THE
(IMP). MYCOPHENOLIC
, THEREBY DEPLETING GUANOSINE MONOPHOPHATE
(GMP), GUANOSINE TRIPHOSPHATE (GTP) AND DEOXYGUANOSINE
distinct mechanisms to reduce the proliferation of T-lymphocytes.
completely dependent on the de
novo guanosine nucleotide synthesis. Second, these cells have a different expression of an
5
Mycophenolate acid mostly influences the interphase of the cell cycle of the T-
lymphocytes, at the level of the G1 phase. That phase is characterized by intensive synthesis
of structural and functional proteins, organelles, and also purines. IMP, XMP, GMP and AMP
are intensively produced. The cell prepares itself for cell division. (FIGURE 1.3.)
FIGURE 1.3.: GENERAL CYCLE OF CELL DIVISION. THE INTERPHASE CONSISTS
OF THREE PHASES, G1 PHASE, S PHASE AND G2 PHASE, FOLLOWED BY MITOSE,
THE ACTUAL CELL DIVISION. THE G1 PHASE IS A MAJOR PERIOD OF CELL
GROWTH. IN THE S PHASE THE CHROMOSOMES BECOME REPLICATED. G2
PHASE IS THE LAST PERIOD DURING THE INTERPHASE, THE CELL UNDERGOES
A PERIOD OF RAPID GROWTH TO PREPARE FOR MITOSIS. TWO EQUAL
DAUGHTER CELLS WITH THE SAME DNA ARE OBTAINED AFTER MITOSIS.
(http://bioinfo.mbb.yale.edu/expression/cluster/cell_cycle.jpg)
1.2.2.2 Effects of MPA on the production of antibodies
In addition to their major role in antibody formation, B-lymphocytes have several
effects on the immune response. They can present antigens to T lymphocytes and contribute
to immunologically driven inflammatory processes. MPA inhibits the proliferation of B-
lymphocytes and the production of antibodies. (C.Allison et al., 2005).
6
1.2.3 Pharmacokinetics of MMF
1.2.3.1 Absorption
After oral administration, mycophenolate mofetil is extensively hydrolysed into its
active form by esterases in the stomach and the small intestine, followed by absorption of
MPA. The enteric-coated mycophenolate sodium is protected from the acid pH in the
stomach. But it is highly soluble in neutral pH at the level of the intestine, which leads to a
greater availability of MPA in the intestine for absorption. (Roche et al.,2009) (Staatz et al.,
2007)
1.2.3.2 Distribution
MPA has a low distribution volume due to strong albumin binding. 97-99% of MPA is
bound to albumin, but it does not bind significantly to the other plasma protein, α1-acid
glycoprotein. The interaction between MPA and albumin does not depend on the
concentration of MPA, but the free fraction can be influenced by the amount of albumin in
plasma. Hypoalbuminaemia can be caused by a liver disease or renal dysfunction. (Roche et
al.,2009) (Staatz et al., 2007)
1.2.3.3 Metabolism
7-O-MPA-glucuronide (MPAG) is the main metabolite of MPA. It posseses no
IMPDH inhibitory activity. Glucuronidation occurs in the gastrointestinal tract, liver and
kidney by uridine diphosphate glucuronosyltransferase (UGT). MPAG can enter into the
enterohepatic recirculation. (Roche et al.,2009) (Staatz et al., 2007)
To a lower extent, two other metabolites are formed: 7-O-glucoside and acyl-MPA-
glucuronide (AcMPAG). 7-O-glucoside has no pharmacological activity, while AcMPAG
appears capable of inhibiting IMPDH. It is also a reactive electrophilic metabolite, which can
cause tissue damage by covalent binding with proteins, lipids and nucleic acids. (FIGURE
1.4.)
7
FIGURE 1.4.: METABOLISME OF MYCOPHENOLIC ACID (Pierrefeu, A. 2007)
1.2.3.4 Elimination
The most important way for elimination of MPAG and AcMPAG is renal excretion
via active tubular secretion (> 90%). 6% of the given dose is excreted by the faeces. Excretion
of MPAG via bilious excretion is also possible, but in smaller amount. (Roche et al.,2009)
(Staatz et al., 2007)
1.2.3.5 Enterohepatic recirculation
MPAG can be excreted into the bile and deconjugated back to MPA by glucuronidase
of bacteria in the colon, thereby allowing that the liberated MPA is absorbed for a second
time, which contributes for 40% of the overall MPA exposure. (Roche et al.,2009) (Staatz et
al., 2007)
8
1.2.3.6 Drug interactions
Interactions were observed with several drugs, such as acyclovir, antacids,
cholestyramine, cyclosporine A, ganciclovir, trimethoprim/ sulfamethoxazole, norfloxacin
and metronidazole. (Roche et al., 2009) (http://www.rxlist.com/cellcept-drug.htm)
1.2.4 Adverse effects
Gastrointestinal and hematological adverse effects as well as vulnerability to
infections are associated with mycophenolate use. Mycophenolate mofetil gastrointestinal
toxicity appears to be dose-related. (Staatz et al., 2007). Patients suffer from nausea,
vomiting, diarrhea and anorexia. More rarely, oesophagitis, gastrointestinal ulcers,
perforations and gastrointestinal haemorrhage are reported. Leucopenia, anemia,
thrombocytopenia are examples of hematological side effects, for which the mechanism
remains unknown. (Shu et al., 2008)
Because of the severe suppression of the immune system, the patient can develop
bacterial, viral or fungal infections, including opportunistic infections, fatal infections and
sepsis. Infections like cytomegalovirus (CMV), candida, pneumonia, urinary tract infection,
herpes zoster, zona, and pancreatitis have been detected.
Another consequence of immunosuppressive therapy is that the patient has an
increased risk for developing malignant carcinomas, like lymphomas and other malignancies,
particularly of the skin. The risk of developing cancer seems to be related to the duration and
intensity of the immunosuppressive therapy.
1.3 INOSINE MONOPHOSPHATE DEHYDROGENASE
1.3.1 Characteristics
Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme in the biosynthesis
of purine nucleotides and an important regulator of cell proliferation. It is a homotetramer
9
with a molecular mass of 56kDa. It catalyzes the oxidation of inosine 5-monophosphate
(IMP) to xanthosine 5’-monophosphate (XMP) and is dependent on nicotinamide adenine
dinucleotide (NAD+) (FIGURE 1.5.) (Qingning Shu et al., 2007).
There are two human isoforms, types I and II, which are derived from different genes,
IMPDH1 and IMPDH2, located on chromosomes 7 and 3. Each type consists of 514 amino
acids with 84% similarity. In most tissues and cell types type I and II IMPDH come to
expression, but a different expression profile is observed in lymphocytes. In mature resting
lymphocytes, IMPDH type I is the dominant species. When the lymphocytes become
activated, IMPDH type II predominates over type I. Both isoforms show similar affinities for
the substrates, NAD+ and inosine 5-monophosphate (IMP) (Sombogaard et al., 2009).
FIGURE 1.5.: THE NOVO BIOSYNTHESIS OF ADENINE AND GUANINE
NUCLEOTIDES AND THE ROLE OF IMPDH. (Shu et al. 2008)
10
1.3.1.1 Mechanism of the conversion of IMP to XMP
“The mechanism of the conversion of IMP to XMP by IMPDH starts with a
nucleophilic attack of an active site cysteine residue to IMP to form a covalent intermediate
(E-IMP*, Fig 1.8). Subsequent hydride transfer to the nicotinamide ring of the cofactor,
NAD+ (electron acceptor), followed by hydration of the resulting intermediate results in the
formation of the tetrahedral intermediate, E-XMP†. The final step is the expulsion of XMP
from the latter intermediate.” (FIGURE 1.6.) (Qingning Shu, 2007).
FIGURE 1.6.: MECHANISM OF CONVERSION OF IMP TO XMP BY IMPDH. (Shu et al.,
2008)
1.3.2 Reaction of MPA with IMPDH
IMPDH posseses three binding sites, an active site for IMP, a cofactor-site for
NAD+/NADH and an allosteric site for specific inhibitors. The third one is a site remote from
the IMP and NAD+ pockets. There are two kinds of IMPDH inhibitors. Competitive
inhibitors, such as the monophosphates and ribavirine, bind to the IMP site and prevent the
natural substrate from binding to the enzyme. It results in a formation of a reversible bond
between IMPDH and the inhibitor. Noncompetitive, reversible IMPDH-inhibitors as MPA
11
interact with nicotinamide adenine dinucleotide (NAD+)-binding pocket and generally show
structural resemblance to NAD+. (Qingning Shu et al., 2008). The mechanism, by which MPA
inhibits the enzyme, appears to be related to the ability of MPA to structurally mimic both the
nicotinamide adenine dinucleotide cofactor and a catalytic water molecule. This prevents the
oxidation of IMP to XMP. (FIGURE 1.7.) (Roche et al., 2008).
FIGURE 1.7.: SCHEMATIC PRESENTATION OF THE INTERACTIONS OF IMPDH
WITH XMP AND MPA.
1.4 DETERMINATION OF THE ACTIVITY OF IMPDH
Therapeutic drug monitoring of MMF is necessary because there exists a wide
interindividual pharmacokinetic variability. The determination can be achieved in two
different ways. Monitoring the plasma concentrations of MPA and MPAG is a possibility, but
may not be sufficient because interindividual differences in IMPDH activity are not taken into
account. (Kamar et al., 2006). Therefore, there is a growing interest to measure IMPDH
activity as a pharmacodynamic parameter. The degree of inhibition of the enzyme gives a
better idea of the MMF-induced immunosuppression. This more advanced, indirect approach
for therapeutic drug monitoring permits to evaluate the immunosuppressive effect of the drug.
(Storck et al., 1999) (Vethe et al.,2006).
12
Several methods are published for determining the activity of IMPDH. Usually the
analyses are carried out with HPLC-UV (Storck et al.,1999) (Khalil et al.,2006) (Albrecht et
al.,2000) (Vethe et al.,2006) (Glander et al.,2009) (Daxecker et al.,2001) (Mino et al.,2009)
(Chiarelli et al.,2010) or LC-MS/MS, which are non-radiolabelled procedures. They are to be
considered as the reference techniques. Possible alternatives for assessing IMPDH activity
include an immunoassay like enzyme multiplied immunoassay technique (EMIT) (Blanchet et
al.,2008) (Van Gelder et al.,2009), and a radiolabeled assay. (Langman et al.,1994).
1.4.1 Radiolabeled assay
Langman et al. use whole blood and isolated lymphocytes for determining the activity
of IMPDH. Briefly, the activity can be determined by measuring radioactive tritium or ³H,
released from tritium labeled hypoxanthine or inosine that is added to the samples before
incubation. An incubation time of 30 minutes was applied. The radioactivity is measured by
scintillation counting. Whole blood was pretreated with heparin, EDTA or acid-citrate-
dextrose anticoagulant. For isolation of the lymphocytes several steps of centrifugation and
washing were applied. (Langman et al., 1995)
1.4.2 Chromatographic methods
1.4.2.1 Preparation of the samples
Depending on the biological matrix, different sample preparation methods have been
reported. The activity of IMPDH can be examined in whole blood (Storck et al.,1999),
erythrocyte lysate (Khalil et al.,2006) )(Mino et al., 2009) or peripheral blood mononuclear
cells (PBMC) (Daxecker et al.,2001)(Albrecht et al.,1999) (Glander et al.,2009) (Chiarelli et
al., 2010) (Cichna et al.,2003). CD4+ cells may also be considered as matrix because
lymphocytes are probably the most important target cells of MPA. (Vethe et al.,2006).
Whole blood samples have to be treated first with an anticoagulant, like heparin
(Maiguma et al.,2010), EDTA (Khalil et al.,2006) (Vethe et al.,2006)(Mino et al.,2009),
13
lithium heparin (Storck et al.,1999)(Albrecht et al.,2000) (Glander et al., 2009) or sodium
citrate (Cichna et al.,2003).
Depending on the biological matrix several ways can be used for further treatment.
PBMC’s or erythrocytes are usually separated by centrifugation. (Glander et al.,2009),
(Daxecker et al.2001), (Albrecht et al.,200) (Mino et al.2009) (Cichna et al.,2003). Vethe et
al., 2006, who determined the IMPDH activity in CD4+cells, isolated the cells from EDTA-
blood sample by using polysterene beads coated with anti-CD4+ monoclonal antibodies.
1.4.2.2 Enzymatic conditions
The IMPDH activity is determined by measuring the formed XMP, after incubation of
the biological sample with IMP and NAD+. Different incubation conditions are summarized in
TABLE 1.1.
1.4.2.3 Chromatographic conditions
Different HPLC methods for determining the activity of IMPDH are summarized in
TABLE 1.2. Besides UV, MS/MS involving an ion trap mass spectrometer with an ESI
(electrospray ionisation) source operating in the negative ion mode can also be used for
detection. (Chen et al., 2009).
Another chromatographic method that can be applied is ion-pair chromatography. In
ion-pair chromatography retention depends on a large number of parameters, including type
and concentration of the ion-pair reagent, pH and ionic strength of the mobile phase,
concentration of the organic modifier or Mg2+, isocratic or gradient elution and column
temperature. (Cichna et al., 2003).
14
TABLE 1.1. ENZYMATIC CONDITIONS
Ref Storage (°C)
Sample volume
Incubation medium Incubation conditions
Termination of the reaction
Post-treatment of the sample
Stor
ck e
t al
.,
1999
-20 1ml of WBC lysate
0.01 ml IMP (0.25 mM) 0.01 ml β-NAD+ (0.25mM)
T = 37°C 60 min
Precipitation by adding 0.15mL 4 M HCLO4
1. Centrifugation 2. Heating 900µL of the supernatant at 100°C, 1 hour. 3. Cooling to room temperature 4. 120 µl of 4 M KOH (pH 5~7) 5. Centrifugation
K
halil
et a
l.,
2006
-21 150µl of RBC lysate
12.5 µl KCl (4M), 12.5µl DTT (40mM), 275µL 0.067M K2HPO4 (pH 7.4) 25µl IMP (10mM) 25 µl β-NAD+ (10mM) Final concentration: 100 mM KCl, 1mM DTT, 0.5mM IMP and 0.5mM β-NAD+
T= 37°C 120 min
Precipitation by adding 25µl cold 60% HCLO4
1. Centrifugation, 4min 2. Salt precipitation with 55µl 5 M K2HPO4, 5min
(adjust the pH to 5.4) 3. Centrifugation
Gla
nder
et
al,
2006
-80 50µl of PBMC lysate
1 mM IMP, 0.5 mM β-NAD+ 40 mM NaH2PO4 (pH 7.4), 100 mM KCl Total volume: 130µl
T = 37°C 150 min
20µl of 4 M HCLO4, placing on ice
1. Centrifugation 2. 10 µl 5 mM K2CO3 3. Storing of the samples for 30 min at -80°C or for 2
hours at -20°C
A
lbre
cht e
t al
., 19
99
-20 1.5 ml WBC fractions
Tris-EDTA-allopurinol buffer 10µl β-NAD+ (final concentration 0.25mM) 10µl IMP (final concentration 0.25mM)
T = 37°C 30 min 60 min
Precipitation by adding 0.15 ml 4 M HCLO4
1. Centrifugation 2. Heated at 100°C, 60 min. 3. Cooling to room temperature 4. 0.7 à 0.9 ml 4M KOH 5. Vortex + centrifugation
Vet
he e
t al.,
20
06
-20 25µl cell-bead suspension
100 µl Tris-EDTA allopurinol buffer IMP: 1.79 µM NAD+ 0.38 µM Total volume: 220 µl
T = 37°C 120 min
Precipitation by placing sample on ice and 32 µl HCLO4 4 M
1. Centrifugation 2. Heating sample at 100°C, 50min 3. Cooling to room temperature 4. 22 µL 4 M KOH 5. Vortex + centrifugation
Min
o et
al.,
20
09
-84 150µl RBC lysate
350µl 50 mM K2HPO4 pH 7.4 50 µmol KCl 0.25 µmol IMP 0.9 µmol NAD+
T = 37°C 180 min
Precipitation by adding 25µl of cold 9.2 M HCLO4
1. Centrifugation at 17.900×g at 4°C, 4m in. 2. Salt precipitation by adding 55µl 5 M K2HPO4 3. Centrifugation at 17.900×g at 4°C, 15min
Chi
arel
li et
al
., 20
10 -80 40µl PBMC
lysate 40mM K2HPO4 pH 7.4 100mM KCl, 1mM IMP, 1mM NAD+
Final volume: 100µl
T = 37°C 180 min
Boiling at 100°C, 10 min. Centrifugation at 12.000×g, 10 min.
15
TABLE 1.2. CHROMATOGRAPHIC CONDITIONS
Ref. Mol added
Volume injection sample
Stationary phase Mobile phase Flow rate (ml/min)
Detection Retention time (min)
LoQ / LoD
Kha
lil e
t al.,
(2
006)
XMP 10 µl supernatant
Hypersyl ODS 125 × 3 mm dp = 5µm 35°C + guard column (80 × 3 mm)
0.025 M Sodium phosphate buffer, pH 5,6 0.025 M TBAHS and ACN Gradient elution: A/B/A Phase A: 1% ACN Phase B: 20% ACN
0.6 UV (254nm)
12
0.5 pmol/µl (LoD)
or 0.5 mmol/l
Alb
rech
t et
al.,
(2
000)
Xanthine 200µl supernatant
Nucleosil C18 150 × 4.6 mm dp = 5µm T = 20 to 25°C
Isocratic conditions 96% water + H3PO4 (pH 1.8) 4% methanol
1.0 UV (260 nm)
22
Vet
he
et a
l.,
(200
6) Xanthine 100 µl
supernatant Nucleosil C18
150 × 4.6 mm dp = 5 µm + guard column
Isocratic conditions 96% water + H3PO4 (pH 1.8) 4% methanol
1.0 UV (260 nm)
20
Gla
nder
et a
l. (2
009)
Center B XMP AMP Center R XMP AMP
Center B 5 µl supernatant Center R 5 µl supernatant
Center B Prontosyl AQ C18 150 mm × 3 mm
dp = 3µm T = 40°c Center R ChromSpher C18
150 × 4,6 mm dp = 5µm T = 40°C
Center B Isocratic conditions 6:94 (v/v) methanol: 50 mM KH2PO4 + 7 mM TBAS (pH 5.50) Center R Gradient elution Phase A: 50 mM KH2PO4 + 7 mM TBAS (pH 5.6) Phase B: methanol
Center B 1.0
Center R
1.0
Center B UV
(256nm)
Center R UV
(254nm)
Center B AMP: 5.9 XMP: 7.6
Center R
AMP: 3.5 XP: 5.6
0.031 µmol/L (LoQ)
or 0.0031 nmol/l
0.010 µmol/l (LoD)
Dax
ecke
r et
al.,
(200
1)
XMP
20µl supernatant
LiChroCART superspher 100 RP- 18 endcapped 250 × 4mm T = 22°C
Phase A: 100 mM H3PO4, pH 6.20 (TEA) Phase B: 100 mM H3PO4, 5mmol/l MgSO4; pH: 6.20 Gradient elution: Initial conditions: 100% (A), 0% (B) Gradient: 3.03% B/min, 33min
1.0 UV (254nm)
9 min
Pate
l, et
al
., (2
007)
XMP 10 µl supernatant
Hypersil ODS-2 150 × 4.6 mm dp = 5 µm T = 30°C
Isocratic conditions 3% Methanol 97% 50 mM KH2PO4 and 7 mM tetra-n-butyl NH4HPO4 (pH 4.5)
0.6 UV (254nm)
1nmol/mL or
1µmol/L (LoQ)
Stor
ck
Et a
l.,
(199
9) XMP 200 µl
supernatant Nucleosil C18 250 × 4.6 mm dp = 5µm T = 20 to 25°C.
Isocratic conditions 4%methanol / 96%water pH = 1.8
0.5 UV (260nm)
18 to 20 min.
16
1.4.3 Conclusion
The determination of IMPDH activity, a pharmacodynamic approach that should
facilitate individualized MMF therapy, is gaining popularity. Several methods have been
developed, but still require further optimization. HPLC-UV and LC-MS/MS are the reference
techniques. Accurate, reliable and reproductive results are obtained, but these techniques are
time consuming. Hence, further investigation is necessary.
17
2 GOAL
Therapeutic drug monitoring of MPA is important, because of the great interpatient
variability in IMPDH activity. By only measuring the plasma concentrations of the drug, it is
not possible to control the therapy. Assessing the IMPDH-activity should facilitate to
optimize the dose of MMF to be administered to the patient to prevent organ rejection.
Obviously a first step is to develop a reliable method to determine the activity of the enzyme.
In this study, Reversed Phase-Liquid Chromatography, followed by UV-detection will
be explored for determining the activity of IMDPH in blood samples. IMPDH converts IMP
to XMP, so by measuring those two components it is possible to determine the activity of
IMPDH. First, the method has to become validated. Selectivity, linearity, accuracy, precision
(repeatability and reproducibility) and limit of quantification (LoQ) have to be investigated.
When the validation meets the requirements, further application for the investigation of the
enzymatic activity of IMPDH can be assessed in biological matrix.
An erythrocyte lysate is chosen as biological matrix in this study. Pretreatment of the
blood will be carried out to obtain the lysate. The production of XMP depends on three
factors, i.e. the incubation time, the concentration of IMP and the concentration of NAD+.
Another goal of this study is to select these parameters for XMP production.
18
3 MATERIALS AND METHODS
3.1 CHROMATOGRAPHIC SYSTEM
In Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) separation
is based on a difference in distribution of substances between two non-miscible phases. The
stationary phase is non-polar and modified silica. A polar aqueous solution is used as the
mobile phase.
The configuration of the HPLC system is summarized in TABLE 3.1.
TABLE 3.1.: DATA OF THE CHROMATOGRAPHIC SYSTEM
Column Nucleodur ® C18 Pyramid, 125 × 4mm, dp: 3µm
Pump 325 system, Kontron ® instruments
Automatic injection HPLC 360 autosampler, Kontron ® intstruments
UV detection HPLC 332 detector, Kontron ® instruments
Software: Goebel instrumentelle Analytik, ® Geminyx Version 1.91
3.1.1 Stationary phase
Nucleodur ® C18 pyramid column was selected as the stationary phase. The silica
phase with hydrophilic endcapping is modified with C18 for 14%. It is stable in 100% aqueous
mobile phase systems and in a pH range from 1 to 9. It has the property to separate very polar
compounds, such as nucleotides by polar interactions (H bonds). In addition, the column
provides the possibility for hydrophobic interaction by the presence of the hydrophobic alkyl
groups C18. By combining those two types of interactions, it can separate the nucleotides in an
acceptable runtime, because they are negatively charged and possess hydrophobic properties.
(http://www.mn-net.com)
The stationary phase possesses a particle size of 3µm, and a pore size of 110 Å. Other
characteristics are that it has a carbon content of 14% and knows a pH stability of 1 to 9. The
dimensions of the column are 125× 4mm.
19
3.1.2 Mobile Phase
The mobile phase consists of a potassium dihydrogen phosphate buffer (KH2PO4;
0.01M; pKa 4.4) adjusted to a pH of 4. Methanol (MeOH) is used as organic modifier. The
isocratic mobile phase consists of a 99:1 (v/v) of 0.01 M KH2PO4 (pH 4) and MeOH. A flow
rate of 0.8ml/min is used.
3.1.3 Detector
UV-detection occurs at a fixed wavelength of 260nm. The system provides a spectrum
of the sample and gives information in two dimensions (time and absorption). The cut-off
values of water (190nm) and methanol (210nm) do not interfere with the UV-absorption. The
mobile phase is compatible with the detector.
20
3.2 MATERIALS
The products used for preparing the samples:
- XMP: Xanthosine 5’-monophosphate dinucleotide disodium salt (Sigma-Aldrich ®)
- IMP: Inosine 5’-monophosphate dinucleotide disodium salt (Sigma-Aldrich ®)
- β-NAD: β-Nicotinamide adenine dinucleotide hydrate (Sigma-ultra ®)
The products used for preparing the mobile phase:
- Methanol (Sigma-Aldrich ®)
- KH2PO4: Potassium dihydrogen phosphate (Merck ®)
- Acetic Acid (Fluka Chemika ®)
- Purified or distillated water
The products used for enzymatic incubation:
- KH2PO4: Potassium dihydrogen phosphate (Merck ®)
- DTT: Dithiothreitol (Sigma-Aldrich ®)
- Versol 0,9% NaCl (Aguettant ®)
- NaOH: Sodium hydroxide (R.P Normapur AR ®)
- 70% HCLO4: 70% Perchloric acid, suprapur (Merck ®)
- KCl: Kaliumchloride (Merck ®)
21
3.3 VALIDATION OF THE METHOD
Validation of the method is required prior to further application. The fundamental
parameters are: selectivity, accuracy, precision (repeatability and reproducibility), linearity
and limit of quantification. It will be discussed in this order below. (Bresole et al., 1996)
(Epshtein et al., 2004)
3.3.1 Selectivity
To confirm the selectivity, it is required that the peaks of the analytes are well
separated from each other and from peaks of the mean impurities (not considered at this
stage). This can be assessed by analyzing a sample that exists of a mixture of IMP, XMP and
NAD+. The selectivity or the separation between two peaks in a chromatogram is expressed
by the resolution (Rs). Rs > 1.5 has to be reached in order to obtain a good selectivity
(Epshtein et al., 2004). The formula to find the resolution is the following:
Rs = (t2-t1) / 0.5 (w1 + w2) (3.1)
Where: t1, t2 = retention times of peak 1 and peak 2
w1, w2 = baseline peak width of peak 1 and peak 2
3.3.2 Linearity
A calibration curve has to be generated for XMP and IMP. By preparing standard
solutions of XMP and IMP, the linearity will be examined. Each solution has to be measured
three times. The response signal Y has to be directly proportional to the concentrations X.
Series of low and high concentrations are used for testing the linearity. The correlation
coefficient r may not be lower than 0.999 for the two regions. (Epshtein et al., 2004)
The concentrations of IMP and XMP tested to assess linearity are given in the
following table (TABLE 3.2.)
22
TABLE 3.2.: TESTED CONCENTRATIONS FOR LINEARITY
IMP XMP
Low concentrations (µM)
High concentrations (µM)
Low concentrations (µM)
High concentrations (µM)
0 5
10 20 30 40 50
50 80 100 150 200 250
0 0.25 0.5 2 5 10
10 20 50 100 150
3.3.3 Accuracy and precision (repeatability and reproducibility)
Accuracy and precision are criteria that are used to express the quality and error of a
method. Three standard concentrations of IMP and XMP that represent the range of both
calibration curves must be used: one concentration, which is more than three times the
concentration determined for LoQ, one average concentration and one high concentration.
The tested concentrations are 0.5µM, 5µM and 20 µM for XMP and 5µM, 20µM and 40µM
for IMP. (Bresole et al., 1996) (Epshtein et al., 2004)
Repeatability is determined on the basis of one day by carrying out a minimum of five
determinations of each standard concentration by the same operator using the same
instrument. By repeating the same analyses over a short period of time and by involving
several analysts the reproducibility can be expressed. The precision around the mean value of
the results has to be evaluated by the calculation of the coefficient of variation (CV) and it has
to be less than 15% for both repeatability and for reproducibility. Accuracy of the results can
be expressed by calculating the percentage error (PE) or percent deviation. The mean value of
the measurements of a standard concentration should be within ±15% deviation of the
theoretical concentration of the standard. When the results meet the requirements, accuracy
and precision is guaranteed for the method. The formulas are given below:
SD = √ ∑ (xi – x)2 (3.2)
n-1
23
VC (%) = SD ×100% (3.3)
x
PE (%) = experimental value – theoretical value ×100 (3.4)
theoretical value
Where: xi = result of concentration (i=1,…n)
x = average concentration
n = number of results
VC = coefficient of variation
SD = standard deviation
PE = percentage error
3.3.4 Limit of quantification
Limit of quantification (LoQ) is the lowest concentration that can be measured with a
good accuracy and precision (repeatability and reproducibility). The variability has to be
investigated as well. By investigating repeatability and reproducibility of the same
concentration the coefficient of variation (CV) can be calculated. It has to be less than 20%.
For accuracy the percentage error (PE) of percent deviation should be ± 20%. (Bresole et al.,
1996) (Epshtein et al., 2004). The concentration tested for LoQ of XMP is 0.125 µM, for IMP
0.250 µM.
It may not be confused with the limit of detection (LoD). This is the minimum
concentration of the substance in the sample that can be detected and that can be distinguished
from the noise level.
24
3.4 INCUBATION CONDITIONS OF THE SAMPLES
3.4.1 Pretreatment of blood samples
The concentration of XMP, formed by the activity of IMPDH, is measured in a lysate
of erythrocytes. Prior to analysis red blood cells were collected and stored for maximum two
days at 4°C.
To prepare the lysate of erythrocytes the following steps have to be carry out: a first
centrifugation has to be done for 15 minutes, at 3500 rpm at 4°C. The supernatant has to be
removed. The precipitation consists of red blood cells and 2ml is used for further treatment.
3ml of cold physiological NaCl solution is added for dilution of the cells, followed by a
second centrifugation during 10 minutes, 3000 rpm at 4°C. The supernatant is disposed and
for a second time 3ml of physiological serum is added. After homogenization 1ml of this
mixture is diluted with 3ml of ice-cold distilled water (1/3 v/v), which causes lyses of the
membranes of the red blood cells. A next centrifugation is performed during 15 minutes, 4000
rpm at 4°C. The supernatant is removed and stored at -20°C in plastic tubes until analyses.
3.4.2 Enzymatic conditions
The reaction environment consists of 100µl of the lysate of the erythrocytes, 100µl of
phosphate buffer (K2HPO4, 0.5M, pH 7.4), 100µl of dithiothreitol (DTT) in phosphate buffer
(K2HPO4, 0.5M, pH 7.4) (1mM DTT), 15µl IMP (1mM) et 15µl NAD+ (0.5mM).
The pH of 7.4 is important for optimal activity of IMPDH. Dithiothreitol (DTT)
possesses reducing characteristics. It protects the enzymatic activity by inhibiting the
formation of intermolecular or intramolecular disulfide bonds between cysteine residues of
IMPDH. The enzyme is present in the lysate of erythrocytes. IMP is the necessary substrate of
the enzyme for formation of XMP. NAD+ is the co-substrate or the cofactor (FIGURE 1.6).
25
3.4.2.1 Incubation conditions
For each sample 100µl lysate is used. After adding 100µl buffer solution, 15µl of a
0.5mM NAD+ solution and 100µl of 1mM DTT solution, the sample is incubated for 3
minutes at 37°C.
The enzymatic reaction is initiated upon addition of 15µl of 1mM IMP solution.
Several incubation times at 37°C are tested: 30, 45, 60, 120 and 180 minutes. The enzymatic
reaction is terminated by adding 10 µl of cold HClO4 70% solution. First, the samples are
cooled by placing on ice, followed by a centrifugation during 15 minutes at 4000 rpm and
15°C. The supernatant after the reaction is recuperated and can be directly used for analysis
with HPLC.
The enzymatic reaction is stopped by adding 70% HClO4. The pH of the reaction
environment decreases to 2. As a result, the enzyme IMPDH becomes denaturized and loses
its activity.
26
4 RESULTS
The following chromatographic conditions are set as a standard in this study. The
range or sensibility can be changed in function of the tested concentration of the nucleotides.
TABLE 4.1.: STANDARD CHROMATOGRAPHIC CONDITIONS
Flow rate: 0.8 ml/min
Injection volume: 80 µl
Mobile phase: 99% K2HPO4 (0.01M) / 1% methanol, pH: 4.00
Pressure: 165 bars
Range: 0.050
4.1 VALIDATION OF THE METHOD
4.1.1 Selectivity
A solution that consists of a mixture of IMP (20µM), XMP (10µM) and NAD+
(25µM) was investigated for selectivity. Using the chromatographic conditions described in
TABLE 4.1. the observed average retention times are 3.10 minutes for IMP, 4.20 minutes for
XMP and 10.30 minutes for NAD+. The RS between IMP and XMP is about 1.2. Despite the
fail that a resolution of 1.2 does not meet the minimal resolution, it was accepted to conserve
the chromatographic conditions in TABLE 4.1. Between XMP and NAD+ RS is more than 4.
The following figure shows a typical chromatogram obtained with this.
FIGURE 4.1.: CHROMATOGRAM OF
IMP, XMP AND NAD+.
4.1.2 Linearity
4.1.2.1 Calibration curve of XMP
Linearity was investigated for XMP by using concentrations from 0.25
(TABLE 3.2).
With linear regression analysis the linearity was calculated for a set of low (0.25
– 2 – 5 – 10 µM) and for a set of high concentration
the whole concentration range
between concentration and AUC is defined by an equation
coefficient. The general equation
27
E 4.1.: CHROMATOGRAM OF THE MIXTURE WITH ORDER OF ELUTION:
Calibration curve of XMP
Linearity was investigated for XMP by using concentrations from 0.25
With linear regression analysis the linearity was calculated for a set of low (0.25
M) and for a set of high concentrations (10 – 20 – 50 – 100
the whole concentration range was checked for valid linearity in general. T
and AUC is defined by an equation and a corresponding correlation
oefficient. The general equation is represented in the following graphic (FIGURE 4
ORDER OF ELUTION:
Linearity was investigated for XMP by using concentrations from 0.25µM to 150 µM
With linear regression analysis the linearity was calculated for a set of low (0.25 – 0.5
100 – 150 µM). Also,
was checked for valid linearity in general. The relationship
and a corresponding correlation
hic (FIGURE 4.2.):
- Low range: y = 5.824 x
- High range: y = 7.848 x
- In general: y = 7.759 x
Where: x = concentration XMP
y = AUC (response)
R2 = correlation coefficient
FIGURE 4.2.: LINEARITY OF XMP
150 µM).
4.1.2.2 Calibration curve of IMP
Similarly, a calibration curve for
With linear regression analysis the linearity was calculated for a set of low (0
– 20 – 30 – 40µM) and for a set of
250µM). Also the general linearity of the whole concentration
AR
EA
28
y = 5.824 x – 0.647; R2 = 0.9989
y = 7.848 x – 16.90; R2 = 0.9997
y = 7.759 x – 7.410; R2 = 0.9996
Where: x = concentration XMP
y = AUC (response)
= correlation coefficient
.: LINEARITY OF XMP OVER A WIDE CONCENTRATION RANGE (0 TO
Calibration curve of IMP
Similarly, a calibration curve for IMP was constructed.
With linear regression analysis the linearity was calculated for a set of low (0
M) and for a set of high concentration (40 – 50 – 60 –
M). Also the general linearity of the whole concentration area was checked. This is
Concentration (µM)
(4.1)
(4.2)
(4.3)
OVER A WIDE CONCENTRATION RANGE (0 TO
With linear regression analysis the linearity was calculated for a set of low (0 – 5 – 10
80 – 100 – 150 –
area was checked. This is
shown in the following graphic. (FIGURE 4
correlation coefficients are:
- Low range: y = 5.697 x + 0.718
- High range: y = 6.620 x
- In general: y = 6.488 x
Where: x = concentration IMP
y = AUC (response)
R2 = correlation coefficient
FIGURE 4.3. : LINEARITY OF IMP
250µM).
AR
EA
29
e following graphic. (FIGURE 4.3.) The equations with the corresponding
y = 5.697 x + 0.718; R2 = 0.9990
y = 6.620 x – 37.74; R2 = 0.9983
y = 6.488 x – 17.82; R2 = 0.9984
Where: x = concentration IMP
y = AUC (response)
= correlation coefficient
: LINEARITY OF IMP OVER A WIDE CONCENTRATION RANGE (0 TO
Concentration (µM)
with the corresponding
(4.4)
(4.5)
(4.6)
A WIDE CONCENTRATION RANGE (0 TO
30
4.1.3 Accuracy and precision (repeatability and reproducibility)
TABLE 4.2. and TABLE 4.3. show the data of repeatability and reproducibility of
XMP and IMP. Precision is expressed as the coefficient of variation (CV), while the accuracy
as percentage error (PE).
TABLE 4.2: RESULTS FOR XMP
Theoretic concentration
(µµµµM)
Experimental average
concentrationa
(µµµµM)
CVb
(%)
PEc
(%)
REPEATABILITY
n = 5
LoQ 0.125 0.135 ± 0.001 1.09 + 8.05
Low standard 0.50 0.47 ± 0.02 4.43 -5.54
Central standard 5.0 4.9 ± 0.2 4.35 - 2.14
High standard 20.0 20.3 ± 0.4 2.12 - 1.46
REPRODUCIBILITY
n = 5
LoQ 0.125 0.135 ± 0.003 1.94 + 8.37
Low standard 0.50 0.50 ± 0.03 6.83 - 0.02
Central standard 5.0 4.9 ± 0.5 9.68 - 2.30
High standard 20.0 20.1 ± 1.60 7.99 + 0.44
a The experimental concentration of each result for each standard is calculated by using the functions of linearity
(see 4.1.2.1), followed by taking the average. The standard deviation is calculated by using formula (3.2). b Coefficient of variation (CV) is obtained by using formula (3.3). c Using formula (3.4) gives the Percentage Error (PE).
31
TABLE 4.3: RESULTS FOR IMP
Theoretic concentration
(µµµµM)
Experimental average
concentrationa
(µµµµM)
CVb
(%)
PEc
(%)
REPEATABILITY
n = 5
LoQ 0.25 0.24 ± 0.02 8.37 - 3.04
Low standard 5.0 5.1 ± 0.30 5.81 - 2.35
Central standard 20.0 20.4 ± 0.169 0.83 - 1.98
High standard 40.0 39.6 ± 0.670 1.69 + 0.953
REPRODUCIBILITY
n = 5
LoQ 0.25 0.22 ± 0.02 9.44 + 14.0
Low standard 5.0 5.2 ± 0.49 9.26 - 4.98
Central standard 20.0 21.8 ± 1.83 8.42 - 8.86
High standard 40.0 41.1 ± 4.08 9.93 - 2.84
a The experimental concentration of each result for each standard is calculated by using the functions of linearity
(see 4.1.2.2), followed by taking the average. The standard deviation is calculated by using formula (3.2). b Coefficient of variation (CV) is obtained by using formula (3.3). c Using formula (3.4) gives the Percentage Error (PE).
32
4.2 ENZYMATIC CONDITIONS
The erythrocyte lysate was prepared from different erythrocyte samples as described
in 3.4.1.
A first assay as described in 3.4.2 was repeated for several days by working with the
same lysate. Several incubation times (30, 45, 60, 120 and 180 minutes) were investigated.
The concentrations used for IMP and NAD+ were 1mM and 0.5mM. A control sample of IMP
(25µM), XMP (10µM) and NAD+ (40µM) was systematically injected in the same run.
The average retention time for each component is the following:
IMP: 3.15min.
XMP: 3.90min.
NAD+: 9.40min.
Same results were obtained for all the different incubation times. No peak was
observed at retention time of XMP. Two peaks were found with retention times
corresponding IMP and NAD+. In the first experiment, a peak was seen at a retention time of
5.20 minutes, but was no longer observed in the following experiments. Regardless of the
incubation times, no formation of XMP could be observed.
4.2.1 Influence of KCl
In a second attempt, we investigated the influence of adding potassium chloride (KCl)
to the incubation medium, on the formation of XMP, as reported by other authors (Khalil et
al.,2006) and (Mino et al.,2009). Two concentrations of KCl were tested: 1mM and 5mM.
The other enzymatic conditions remained the same. An incubation time of 2 hours was
applied. Unfortunately no peak of XMP could be detected.
33
4.2.2 Influence of HClO4
The stability of XMP in an acid environment, obtained by adding 70% HClO4, was
investigated by preparing a solution of XMP (20µM) and a solution of XMP (20µM)
containing 70% HClO4. No difference was observed on both chromatograms. XMP was
detected at a retention time 3.90 minutes and no peak for possible degradation products of
XMP were observed.
4.2.3 Assays of previously reported publications
These results forced us to analyze a different erythrocyte. The same enzymatic and
chromatographic conditions (TABLE 4.1) were used. An incubation time of 2 hours was
applied. However again, no XMP could be detected. No peak was observed at the retention
time of 3.90 minutes. However, a peak with a retention time of 5.20 minutes was observed.
Subsequently we proposed to test the methods described in the literature (Khalil et al.,
2006) and (Mino et al., 2009). The enzymatic conditions are described in TABLE 1.2.
Incubation times of 2 and 3 hours were implied. The method of Khalil et al., 2006 uses KCl
(100mM) and DTT (1mM) in the incubation, while Mino et al., 2009 add only KCl (100mM)
to the incubation medium. The method is this study uses DTT (1mM) in the incubation. For
all the three methods, which were carried out simultaneously, no peak was observed at the
retention time of XMP, but the peak at 5.20 minutes was found.
FIGURE 4.4.: CHROMATOGRAM OF THE GENERAL METHOD (3
INCUBATION TIME OF 2 HOURS AT 37°C WAS APPLIED. THE PEA
RETENTION TIMES OF IMP AND
SEEN, BUT AN UNKNOWN PEAK AT 5.20 MINUTES IS
4.2.4 Influence of the pretreatment of the erythrocyte lysate
The influence of the preparation of the erythrocyte lysate
method for preparing the lysate is comparable with the method
The difference with Mino is that a DTT solution (5mmol/L) was used for haemolysis of the
erythrocytes for direct protection of the enzyme IMPDH. In all assays performed, no peak
was formed at the retention time of XMP, while a peak at a retention time of 5.20 minutes
was observed on the chromatograms.
The influence of the dilution of the lysate during the pretreatment step was tested.
Erythrocyte lysate was prepared in the proportion 1/1 (v/v) 34
ATOGRAM OF THE GENERAL METHOD (3
INCUBATION TIME OF 2 HOURS AT 37°C WAS APPLIED. THE PEA
RETENTION TIMES OF IMP AND NAD+ ARE OBSERVED. NO PEAK OF XMP WAS
UNKNOWN PEAK AT 5.20 MINUTES IS FOUND.
Influence of the pretreatment of the erythrocyte lysate
The influence of the preparation of the erythrocyte lysate was also investigated.
method for preparing the lysate is comparable with the method applied by Khalil
is that a DTT solution (5mmol/L) was used for haemolysis of the
erythrocytes for direct protection of the enzyme IMPDH. In all assays performed, no peak
was formed at the retention time of XMP, while a peak at a retention time of 5.20 minutes
rved on the chromatograms.
The influence of the dilution of the lysate during the pretreatment step was tested.
Erythrocyte lysate was prepared in the proportion 1/1 (v/v) so that a higher
ATOGRAM OF THE GENERAL METHOD (3.4.2). AN
INCUBATION TIME OF 2 HOURS AT 37°C WAS APPLIED. THE PEAKS WITH
OBSERVED. NO PEAK OF XMP WAS
was also investigated. The
applied by Khalil and Mino.
is that a DTT solution (5mmol/L) was used for haemolysis of the
erythrocytes for direct protection of the enzyme IMPDH. In all assays performed, no peak
was formed at the retention time of XMP, while a peak at a retention time of 5.20 minutes
The influence of the dilution of the lysate during the pretreatment step was tested.
so that a higher concentration of
the enzyme in the reaction environment w
XMP was seen. A peak at 5.20 minutes was observed.
4.2.5 Blank samples
Finally, blank samples were prepared without adding IMP
using also the method of Khalil and Mino. One blank sample was incubated for 2 hours at
37°C, another blank sample was not incubated.
of 5.20 minutes was observed, but
again a peak at 5.20 minutes (FIGURE 4.5)
FIGURE 4.5.: CHROMATOGRAM OF A BLANK SAMPLE,
STANDARD CONDITIONS
AND XMP ARE OBSERVED. AN UNKNOWN PEAK AT 4.95
PEAK AT A RETENTION TIME OF
35
the enzyme in the reaction environment was achieved. Still no peak at a retention time
5.20 minutes was observed.
blank samples were prepared without adding IMP to the incubation medium,
the method of Khalil and Mino. One blank sample was incubated for 2 hours at
37°C, another blank sample was not incubated. In the latter case no peak with a
of 5.20 minutes was observed, but the chromatogram of the incubated blank sample sho
(FIGURE 4.5).
.5.: CHROMATOGRAM OF A BLANK SAMPLE, OBTAINED UNDER
S (3.4.2). NO PEAKS WITH RETENTION TIME
BSERVED. AN UNKNOWN PEAK AT 4.95 MINUTES AND
PEAK AT A RETENTION TIME OF NAD+ ARE SEEN.
a retention time of
the incubation medium,
the method of Khalil and Mino. One blank sample was incubated for 2 hours at
In the latter case no peak with a retention time
m of the incubated blank sample showed
OBTAINED UNDER
TENTION TIMES OF IMP
MINUTES AND THE
36
5 DISCUSSION
5.1 VALIDATION OF THE METHOD
During this study the validation of an RP-HPLC method was examined. It shows
selectivity, linearity, precision and accuracy.
An acceptable selectivity was obtained (see 4.1.1). IMP shows the highest polarity,
followed by XMP and NAD+. The Rs between IMP and XMP, the most closely spaced
components, is about 1.2 and does not meet the requirement of Rs > 1.5. However, the
separation between the two components is acceptable. The RS between XMP and NAD+ is
more than 4.
Linearity of XMP and IMP was analyzed for two series of low and high concentrated
dilutions. The R2 for XMP, both for the low and high concentration range, is higher than
0.999. It can be concluded that the response signal Y is proportional to the concentrations of
XMP (FIGURE 4.2.). Similar results were obtained for IMP. The R2 of the low concentration
area is 0.999. For the higher concentrations and in general the R2 is 0.998. The response
signal Y is proportional to the concentrations of IMP. (FIGURE 4.3.) The generated equations
can be used for calculations of the accuracy and precision.
The conditions for precision (repeatability and reproducibility) are acceptable for
XMP and IMP. By utilizing the equations the concentrations can be calculated for each
response Y (AREA) of the standards. The precision for repeatability, determined in one day,
has a coefficient of variation (CV) less than 15% for all concentrations tested (0.5, 5, 20µM
for XMP and 5, 20, 40µM for IMP). The precision is also valid for LoQ since CV does not
exceed the 20%. The same can be concluded for the precision of the reproducibility. The CV
does not exceed the given value of 15% for the three standard concentrations, nor the 20% for
LoQ.
The accuracy criteria, defined by the percentage error (PE), are met for all standard
concentrations of XMP and IMP tested, even for their LoQ (<15%).
37
The LoQ of XMP is 0.125µM, which is comparable with the LoQ obtained by (Patel
et al., 2007).
As general conclusion we can state that the validation of our RP-HPLC method was
acceptable with regard to linearity, accuracy and precision, despite a low selectivity.
5.2 ENZYMATIC CONDITIONS
Some hypotheses were proposed to explain the lack of formation of XMP by IMPDH.
The storage of the erythrocyte lysate at -20°C during several weeks could affect the activity of
IMPDH in the erythrocytes. However Glander et al., 2009 have published that lysate samples
could be stored at -20°C and -80°C for 6 months.
The stability of XMP is not influenced by addition of 70% HClO4. We demonstrated
that XMP remains stable in the acidic environment. KCl does not seem to influence the
activity of the enzyme.
In the several assays with the three different methods (3.4.2) (Khalil et al,. 2006) and
(Mino et al., 2009) the results were the same using to two different erythrocyte samples. A
great peak is detected with a retention time of 5.20 minutes, regardless the presence or
absence of KCl or DTT. This peak shows a higher response when a 1/1 (v/v) dilution of the
lysate was used. Different hypotheses may be suggested. It could be a substance of the
biological matrix that is detected at 260 nm. It can also point to the formation of a substance
during the incubation, because other enzymes are present in the biological matrix. Since XMP
can be further transformed to GMP by GMP synthetase (see figure 1.5) (Shu Q. et al., 2008),
a solution of guanosine 5’monophosphate (GMP) was prepared for comparison. However
GMP shows a retention time of 3.30 minutes; and thus the unknown peak of 5.20 minutes
cannot be attributed to GMP. Remarkably, the same peak was observed in blank samples.
Another possible explanation for the lack of XMP formation could be sought in the
preparation procedure of the lysate. However, the procedure used is essentially the same as
38
the ones reported by (Khalil et al., 2006) and (Mino et al.,2009). The difference is that (Mino
et al.,2009) used a DTT-solution (5mM) for immediate protection of the enzyme during
haemolysis of erythrocytes. However, in our hands the incorporation of this procedure did not
significantly influence the outcome of the enzymatic reaction.
The lack of XMP formation could also be caused by a low activity of the enzyme
itself. Glander et al., 2006, published a statistical distribution of the activity of IMPDH and
showed a higher proportion of patients with a lower activity of IMPDH. The blood samples
that were used in this study could coincidently contain low levels of IMPDH.
39
6 CONCLUSION
During this study validation of an RP-HPLC method was investigated. Selectivity,
linearity, accuracy, precision (repeatability and reproducibility) and limit of quantification
(LoQ) for both substances IMP and XMP measure up to the prescribed requirements. The
method is valid for further application to determine the activity of IMPDH in erythrocyte
lysate.
Several incubation assays on different erythrocyte lysates were performed. The
influence of KCl, HClO4 and pretreatment of the lysate were investigated. Also the
procedures of (Khalil et al.,2006) and (Mino et al.,2009) were applied. Still no formation of
XMP could be observed by HPLC. A peak with a retention time of 5.20 minutes was seen
during several assays, also with blank samples after incubation. This could point to another
active enzymatic system in the erythrocyte lysate, which is unknown. In general, it can be
concluded that further investigation on the incubation conditions is necessary.
40
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