Assaying of Warfarin in Iranian Warfarin Resistance Patients Blood by HPLC
A simple and rapid HPLC method with UV detecting system
has been used in determination of warfarin level in plasma of Iranian
patients who received different doses of this drug. Six resistance (10-70
mg day-1) and 5 sensitive patients (0.5-2.5 mg day-1)
were selected for this study. Range of warfarin level in plasma was between
0.93 and 22.8. After determination of warfarin level in warfarin sensitive
and especially, warfarin resistance patients, we are going to find a relationship
between this level and pharmacokinetic or pharmacogenetic factors. In
the separate study which was done in our laboratory on the gene that is
possibly responsible for warfarin resistance we did not find any mutation
in our patient with high warfarin concentration in their blood.
Warfarin is one of the most widespread oral anticoagulant
drug employed (Hirsh et al., 2003). However, its required dose
is highly variable both inter-individually and inter-ethnically (Zhao
et al., 2004). The result of this variability is that some patients
require less warfarin dose than others, warfarin sensitive patients and
some of them need more warfarin dose, warfarin resistance patients. This
sensitivity or resistance to warfarin is due to pharmacogenetic events
in individuals. Warfarin sensitivity is due to the mutation in CYP2C9
gene (Gage et al., 2004). This gene is a liver enzyme required
for oxidation of a large number of drugs including warfarin (Rettie et
al., 1992). Very recently, a novel gene responsible, at least in part,
for the activity of the Vitamin K Epoxide Reductase (VKOR) complex has
been identified (Rost et al., 2004; Li et al., 2004). This
gene is VKORC1 that is the subunit of VKOR and believed that its mutation
is responsible for resistance to warfarin. VKOR is one of the component
of the vitamin K cycle and the therapeutic target site of warfarin.
Because of this variability in warfarin's pharmacokinetic
properties, physicians have to discover the proper maintenance dose by
trial-and-error. Bleeding or other diverse events is the most serious
complication of warfarin (Bogousslavsky and Regli, 1985; Landefeld and
Beyth, 1993; Gullov et al., 1994). So, much effort has been devoted
to improve the safety of this drug by predicting warfarin dose in patients
before treatment. Since different factors may influence the kinetic and
pharmacodynamic of warfarin, Prothrombin Time (PT) and International Normalize
Ratio (INR) is not sufficient for monitoring the oral therapy of warfarin
and different kinds of analytical methods have been developed to determine
concentration of this drug in biological fluids (Helford, 1986; Chan and
Woo, 1988). The aim of this study is to set up a HPLC technique to determine
warfarin concentration in the plasma of Iranian patients who received
different doses of warfarin. In this way, we can find whether resistance
to warfarin is due to pharmacogenetic factors, or pharmacokinetic of this
drug in blood.
MATERIALS AND METHODS
Chemicals and reagents: Warfarin standard was purchased from Sigma
(St. Louis, Mo, USA), HPLC grade methanol, acetonitrile were obtained
from E. Merck (Darmstadt, Germany). Deionized water was used for preparation
and dilution of all solutions.
Apparatus: The HPLC system consisted of a HPLC pump, an injector,
variable wavelength UV detector with an integrator all from waters and
stainless steel reverse phase (C18) column (4 mm
ID, particle size 5 µm) (Knauer). In this system a mixture of filtrate
and degassed 10 mM phosphate buffer (pH: 3.5), methanol and acetonitrile
in 53:7:40 ratio was used as a mobile phase. We also adjust the flow rate
on 1 mL min-1, temperature on 25°C; UV detector on 270 nm,
injection volume is 20 µL and detector range on 0.001 aufs.
1-5: Warfarin sensitive, 6-11: Warfarin resistant
*: F: Female, M: Male, **:
Prothrombin Time, ***: International Normalize Ratio
Patients: Iranian patients, who were prescribed oral warfarin
either at high or low dose, were recruited from Imam Hospital (Tehran,
Iran). Approximately 5 mL of blood sample were collected from volunteers
in a tube containing 5 mg of EDTA. Plasma PT determination and complete
clinical summary of all patients were obtained by clinical staff of the
hospital (Table 1).
Procedure: The analyzed sample was prepared by precipitation method.
In which, to 500 µL of plasma in a tube, 1 mL of methanol was added. The
mixture was vortex for about 1 min to obtain a complete homogenous mixture.
The tube was tightly closed and centrifuged for 10 min at 8000 rpm. Supernatant
layer was separated and centrifuged two more time until we obtained a
clear solution. Twenty microliter of this solution was injected into the
RESULTS AND DISCUSSION
High Performance Liquid Chromatography (HPLC) is one of
the most popular methods for determination of concentration of coumarin
type drugs like warfarin. Its facilitate analysis as well as increasing
sensitivity and resolution are the most advantages of HPLC technique.
In this way, several HPLC methods with UV detectors were described for
determination of warfarin concentration in blood (De Veries and Volker,
1990; Lotfi et al., 1996; Andalibi et al., 1998). Therefore,
we use this method for present study.
In this study, we examined six warfarin resistant patients,
that their warfarin dose is more than 10 mg day-1. We also
examined five warfarin sensitive patients, as control group, that use
less than 5 mg of warfarin per day. None of them use any kind of interfering
drugs. Plasma sample of these individuals were prepared as described earlier.
Chromatograms obtained with
the blank (A) and the standard at 5 µg mL-1 warfarin
concentration (B). The numbers above the peaks correspond to
the retention time. X-axis is time (min.) and Y-axis is absorption
Chromatograph peaks of extracted blank plasma and blank
Plasma spiked with warfarin were well separated (with 6.29 min retention
time of warfarin) and didn`t show any interference (Fig.
1). Five known warfarin concentration ranging from 0.5-25 µg L-1
were analyzed. They yielded a straight calibration curve with r2
= 0.9991 when peak height was plotted against the concentration of warfarin
(Fig. 2). In this way warfarin concentration was determined
for all of the patients and the results are shows in Table
Andalibi et al. (1998) determined warfarin dosage
requirement in Iranian patient. They found that their therapeutic level
is 1193.81±300.35, i.e., they are more sensitive to warfarin than North
American and European patients. Therefore, our five sensitive patients
(control group), who have warfarin level between 0.8-1.18 mg L-1,
are in the normal therapeutic range. However, interesting results were
shown in warfarin resistance patients. Three of them, which use 10-15
mg of warfarin per day, have blood warfarin level in therapeutic range
too, but three other one (two of them need 15 mg and the third needs 100
mg of warfarin per day), showed blood warfarin level more than 2 mg L-1.
These differences in required dose of warfarin resistance
patients, suggest differences in pharmacokinetic of warfarin. In the first
three patients, pharmacokinetic events are responsible for reduction of
warfarin concentration in blood. Therefore, in order to
Warfarin calibration curve;
equation describing the relationship, y = 3.9795x-0.5594; r2
||Blood warfarin concentration in individuals
reach its therapeutic range, they need more warfarin dose.
However, in the other three patients increasing warfarin dose, increase
its concentration in blood. Therefore, they have normal warfarin metabolization
and other reasons such as low receptor activity, or pharmacogenetic effects
are responsible for their high required warfarin dose. The last is may
be due to mutation in VCORC1 gene, which is located on the short arm of
chromosome 16 and now has been identified as therapeutic target site of
warfarin (Rost et al., 2004; Li et al., 2004).
Further work in our laboratory to determine the exact role
of pharmacogenetic factors in warfarin resistance Iranian patients shows
that these patients have no mutation in their VKORC1 gene. We suggest
that other genes may responsible for warfarin resistance in Iranian patients.
The authors wish to thank Imam Hospital and Dr. M. Amanlou
for their grateful support of this research.
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