university of ghent faculty of pharmaceutical...

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





Professeur R. Boulieu

Academic year 2009 – 2010



ERYTHROCYTE LYSATE.


Kirsten VANDERCRUYSSEN

First master in drug devoloppment

Promoter

Prof.Dr. S. Van Calenbergh

Commisaris

Dr. K. Boussery

Prof. T. De Beer








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


4.1.1 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


5.2 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

http://www.fk.cvz.nl/

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)


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.


(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)


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.


coated mycophenolate sodium or Myfortic ®, a

of the active substance. (Glander et


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


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


(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


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

http://www.rxlist.com/cellcept-drug.htm

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.

http://www.mn-net.com/

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


4.1.1 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


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


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


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



rved on the chromatograms.


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




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


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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