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OUTLINE

 1. Abstract
 2. Keywords
 3. 1. Introduction
 4. 2. Materials and methods
 5. 3. Results and discussion
 6. 4. Summary and conclusion
 7. Declaration of Competing Interest
 8. Acknowledgements
 9. References

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FIGURES (1)

 1. 




TABLES (2)

 1. Table 1
 2. Table 2




JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS OPEN

Volume 1, June 2023, 100007



COMPARISON OF THE USE OF 6-THIOGUANINE RIBOSIDE VERSUS 6-THIOGUANINE AS
CALIBRATION STANDARD TO MONITOR 6-THIOGUANINE NUCLEOTIDES IN RED BLOOD CELLS

Author links open overlay panelRoselyne Boulieu a b, Antoine Tourlonias a b,
Magali Larger a c d
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ABSTRACT

The analytical methods reported to evaluate 6-Thioguanine nucleotides (6-TGNs)
level in red blood cells (RBC) are based on the conversion of 6-TGNs to
6-Thioguanine (6-TG) using 6-thioguanine as calibration standard.

Using the LC-DAD method previously reported by Dervieux and Boulieu in 1998 to
determine 6-TGNs, we evaluated the use of 6-Thioguanine Riboside (6-TGR) as
standard instead of the base 6-TG for the monitoring of 6-TGNs in RBC from
patients with IBD.

Our results show that 6-TGN values measured in RBC from 30 patients were
significantly (p < 0,00001) higher when 6-TGR was used as calibration standard
compared to 6-TG. The difference observed may be explained by the presence of
ribose in the chemical structure of 6-TGR contrary to 6-TG. This difference in
6-TGN values was also observed in external quality control assay using 6-TGR
versus 6-TG calibration standard.

The use of the nucleoside 6-TGR as calibrator constitutes a better reflection of
the chemical reaction which occurs in RBC compared to 6-TG. This preliminary
observation suggests that the choice of calibration standard to monitor 6-TGNs
may have a significant impact on the values measured in patients and laboratory
should be aware of this potential pitfall.

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KEYWORDS

Thiopurine
6-TGN
Drug monitoring
HPLC
IBD


1. INTRODUCTION

Azathioprine (AZA) and its metabolite 6-mercaptopurine (6-MP) are the 2 major
immunomodulators that are increasingly used in the treatment of inflammatory
bowel disease (IBD) [1]. AZA, an inactive prodrug, is converted into 6-MP and
then metabolized by a multi-enzymatic process to produce the active
6-thioguanine nucleotides (6-TGNs). The 6-TGN can be incorporated into DNA or
RNA to inhibit replication, DNA repair mechanisms, and protein synthesis, and is
recently reported to inhibit the Rac1 protein to induce T-cell apoptosis by
blocking CD28 signaling. The 6-TGNs are believed to be responsible for
antimetabolic effects [2] and cytotoxicity [3]. Competitively, thiopurine
methyltransferase (TPMT) converts AZA/6-MP into the 6-methyl-mercaptopurine
nucleotides (6-MeMPN), which are associated with hepatotoxicity [4] and
myelosuppression [5]. Therefore, assessment of TPMT phenotype or genotype before
initiating thiopurine therapy may help to prevent severe myelotoxicity following
standard dose of thiopurine.

Steady state 6-TGN concentrations are typically reached within 3 weeks after
initiation of AZA therapy and are commonly used as a surrogate marker for
therapeutic efficacy and toxicity [1], [6]. In contrast, some studies failed to
find any relationship between 6-TGN concentration and therapeutic response [7],
[8].

Among the different HPLC methods developed to determine 6-TGN levels in RBCs
(Lennard, Dervieux, Vikingsson [9], [10], [11]), two methods were widely used
(Lennard and Dervieux) with a safer and simple sample preparation for the method
reported by Dervieux due to the lack of use of toluene as extraction solvent
[12].

The principle of the assay is based on the conversion of 6-TGNs into the base
6-thioguanine (6-TG) by acid hydrolysis, in the presence of perchloric acid and
heat. In the original method reported by Dervieux & Boulieu, the standard used
for calibration is 6-TG. This choice was justified by the lack of availability
of commercial nucleoside and nucleotide of 6-thioguanine in 1998. With regard to
the mechanism of the acid hydrolysis and the availability of 6-thioguanine
riboside, we decided to change the standard used for calibration and replace
6-TG by 6-TGR. The aim of this study was to evaluate the influence of this
modification on the drug monitoring of RBC 6-TGN in patients treated by AZA for
IBD.


2. MATERIALS AND METHODS


2.1. REAGENTS AND STOCK SOLUTIONS

2-amino-6-mercaptopurine (6-TG), 2-amino-6-mercaptopurine riboside (6-TGR) and
DTT were obtained from Sigma. Methanol, potassium dihydrogenophosphate,
chlorhydric acid and perchloric acid were obtained from Merck. Stock solutions
were prepared in 0.1 mol/L HCl and stored at −80 °C. All solutions were stable
at −80 °C for 4 weeks.


2.2. APPARATUS AND CHROMATOGRAPHIC CONDITIONS

The liquid chromatograph consisted of a model 510 pump connected with a
photodiode array detector model 960 and Wisp 715 solvent delivery model (all
from Waters).

The method of Dervieux and Boulieu [10] was used to determine thiopurine
concentrations. Briefly, 6-TG liberated after hydrolysis was analyzed by a
reversed-phase HPLC method. The separation was performed on a Purospher RP 18-e
guard column (from Merck) with a linear gradient elution mode with 0.02 mol/L
potassium phosphate (pH 3,5) and 0.02 mol/L potassium phosphate (pH3,5):methanol
(40:60 by vol). The concentration of methanol varied from 0 to 200 mL/L over a
period of 12 min. The flow rate was 1.2 mL/min. Detection of 6-TG was performed
at 341 nm. Peak identity was confirmed through library matching by comparison of
unknown peak to reference spectra of calibrator. All analyses were performed at
ambient temperature. The Purospher RP 18-e column has demonstrated a long
lifetime:> 500 samples were injected into the column without any deterioration
of its performance.


2.3. SAMPLE COLLECTION AND STORAGE

Blood samples (5 mL) were collected into heparinized tubes and centrifuged
without delay at low temperature (4 °C). Hematocrit were obtained from each
sample. Plasma, leukocytes and the upper layer of RBCs were removed. RBCs were
washed once with isotonic NaCl. After centrifugation for 10 min at 2000g, an
aliquot of packed red blood cells were diluted in isotonic NaCl to determine the
number of red blood cells using automatic counter. The remaining packed RBCs
were stored at −80 °C until analysis. Drug concentrations were normalized to
8.108 RBCs.

The samples used for the comparison are lysates stored at −80 °C less than 3
months from IBD patients treated with azathioprine whose metabolites had already
been determined by the laboratory using 6-TGR as substrate. The values obtained
by the laboratory using 6-TGR as standard are considered as reference
concentrations.


2.4. SAMPLE TREATMENT

250 μL of the RBCs were transferred into a tube, 250 μL of DTT (20 mg/mL) were
added and the mix was rapidly deproteinized by 50 μL of 65% perchloric acid. The
deproteinized samples were centrifuged at 4000g for 15 min at 4 °C. The acid
supernatants were transferred into tubes and centrifuged at 13,000g for 5 min at
4 °C. The acid extracts were transferred into a tube and then heated for 45 min
at 100 °C to hydrolyze thiopurine nucleotides into their bases. After cooling, a
100 μL aliquot was injected into the column. All assays were run in duplicate.


2.5. CALIBRATION CURVES AND CONTROLS

Calibration curves were prepared by adding known amounts of 6-TG or 6-TGR to a
pool of red blood cell hemolysates isolated from blood bank samples from healthy
blood donors. The calibration curves were constructed with 6-TG or 6-TGR
concentrations of 0.6, 1.5, 3.0, 6.0 and 8.0 nmol/mL of red blood cells. Red
blood cell hemolysate was homogenized in a 1.5-mL polypropylene tube with 300 μL
of water containing 0.2 mol/L DTT and the totality was rapidly deproteinized by
50 mL of 700 mL/L perchloric acid. The deproteinized samples were centrifuged at
3000g for 15 min at 4 °C. The supernatants were removed and the acid extracts
were then heated for 45 min at 100 °C to hydrolyze thiopurine nucleotides into
their bases. After cooling, a 80-μL aliquot was injected into the column.

Three controls levels of 2.0, 4.0 and 6.0 nmol/mL using 6-TG or 6-TGR in red
blood cells were prepared according to the sample treatment procedure described
above, each control was assayed in duplicate. Of the three levels, if two
deviated more than 10% from the theoretical values, the calibration curve and
corresponding samples were excluded.


2.6. RBC LYSATES OF IBD PATIENTS

Patient samples for whom a request for monitoring of thiopurine metabolites has
been prescribed as part of conventional clinical follow-up were used for quality
laboratory study. The study was conducted in accordance with the Basic &
Clinical Pharmacology & Toxicology policy for experimental and clinical studies
[13]. According to French legislation, no submission to ethic committee was
required for quality laboratory studies. 39 RBC samples from IBD patients were
analyzed using 6-TG and 6-TGR calibration curves. Sample treatment procedure of
RBC lysates was the same as those described above. 5 RBC lysates were excluded
due to controls out of limits and 4 because of a difference between our
measurement and the laboratory measurement greater than 35%.


2.7. EXTERNAL QUALITY CONTROL

An external quality control program for thiopurine nucleotides was proposed by
SKML society (www.skml.nl). RBC lysates samples were sent to international
laboratories which subscribed to this program. A comparative assay was performed
in our laboratory as follow: RBC lysates (n = 6) from SKML, were analysed using
both 6-TG as calibration standard conformly to our original method published in
Clinical Chemistry in 1998 and considered as one of the international reference
method and also 6-TGR as calibration standard. The data were compared to the
target value of our original method.


2.8. STATISTICAL ANALYSIS

Shapiro-Wilk test was used to evaluate normal distribution and the paired t-test
was used to compare the data obtained with both standards.


3. RESULTS AND DISCUSSION

The calibration curves were linear, with correlation coefficients higher than
0.997 for both 6-TG and 6-TGR. The typical regression equations were y = 26179x
+273.15 for 6-TG and y = 17904x +618.17 for 6-TGR. The coefficient of variation
for control ranged from 3%, 6% and 11% for 6-TG levels of 6.0, 4.0 and 2.0
nmol/mL respectively and from 5.0%, 7.0% and 9.0% for 6-TGR levels of 6.0, 4.0
and 2.0 nmol/mL respectively.

Fig. 1 shows the relation between the reference concentrations previously
measured by the laboratory in the framework of drug monitoring of patient
samples and the measured concentrations obtained in the current assays. The
reference concentrations were measured using 6-TGR as standard following a
modification of the original method (10) previously reported by Dervieux and
Boulieu in our laboratory. The top line corresponds to the values obtained using
6-TGR, with a slope of 1.0 which are in agreement with the results previously
determined. The bottom line corresponds to the results obtained using 6-TG and
exhibits a slope of 0.8. These data demonstrate that the use of 6-TGR compound
as standard leads to a higher value compared to the use of 6-TG compound. For
the same samples, the original method using 6-TG as standard detect 0.809 times
fewer compounds than the new method using 6-TGR. The study of the correlation
between the two methods shows a difference of a factor of 1.24. It means that
the 6-TGN concentrations were 1.24 higher when 6-TGR is used compared to 6-TG.

 1. Download : Download high-res image (140KB)
 2. Download : Download full-size image

Fig. 1. Monitoring of the 6-TGNs concentration from patient samples using 6-TG
and 6-TGR standard compared to the reference concentration.

The Shapiro-Wilk test shows that the variables from which our patient samples
were drawn follow a normal distribution with a 5% risk of error.

The paired t-test was used to assess whether there is a difference between the
mean values of 6-TG and 6-TGR. Table 1 shows the statistical analysis of the
paired t test: the p value< 0.001 means that the difference between the two
methods is significant with a 5% risk of error.

Table 1. Results of paired t-test.

Mean difference1.235t (test value)7.760t (critical value)2.045dF29p-value (two
tail)<0.0001

Table 2 exhibits the comparative data obtained between the use of 6-TG or 6-TGR
as calibration standard for the thiopurine external quality control. As
observed, the values corresponding to the use of 6-TG standard ranged from 7.4%
to 22.0% of the target values evaluated from16 different laboratories using our
original method published in 1998 while the values corresponding to the use of
6-TGR standard ranged from 24% to 56% of the target values. These results
confirmed the influence of the calibration standard on the 6-TGN values
calculated. Otherwise, a wide variability in 6-TGN values was observed in the
data reported by the different laboratories involved in the external quality
control program for thiopurine nucleotides (data not shown).

Table 2. 6-TGN levels (nmol/mL) determined using 6-TG or 6-TGR as calibration
standard compared to target values obtained from external quality control.

6-TGN Target value *6-TGN level /6-TG standard (% target value)6-TGN level/6-TGR
standard (% target value)1.211.30 (7.4%)1.50 (24.0%)2.833.20 (13.0%)3.70
(31%)1.742.10 (21%)2.37 (36%)3.484.10 (18%)4.58 (32%)4.525.53 (22%)7.06
(56%)1.641.94 (18%)2.48 (51%)



mean value using Dervieux et al. method (Clin Chem 1998) obtained from 16
different international laboratories.



Our study shows that the 6-TGN values obtained from IBD patients were
significantly higher when 6-TGR is used as standard compared to 6-TG. This
observation may be explained by the physicochemical structure of the compounds.
6-TG corresponds to the purine base and 6-TGR corresponds to the nucleoside
which included a ribose moiety. The principle of the preanalytical process
previously reported [10] is based on an acid hydrolysis at 100 °C of
6-Thioguanine Nucleotides (6-TGNs) which leads to the liberation of the purine
base by cleavage of the covalent bound between the base and the ribose.
Considering the structure of 6-Thioguanine Riboside (6-TGR) including a ribose,
the reaction which occurred during acid hydrolysis is similar to the reaction
that occurs with 6-TGN. While when 6-TG is used as standard, the cleavage of the
link between the base and the ribose is not possible due to the lack of ribose
compound.


4. SUMMARY AND CONCLUSION

The present study demonstrates that 6-TGN values measured in RBC from IBD
patients were significantly higher when 6-TGR was used as calibration standard
compared to 6-TG.

With regard to the difference observed in the 6-TGN values in function of the
standard used, a potential clinical impact exists. Considering the therapeutic
values recommended (250–450 pmol/8.108 RBCs) [14], [15], [16] for 6-TGNs and the
potential risk of side effects, the data reported need to be taken into account
to avoid the following situations:

1/The use of 6-TG as standard gives a 6-TGN concentration below the therapeutic
values while the use of 6-TGR standard gives value in the therapeutic range.
This can lead to an unnecessary increase of AZA dose with a risk of side
effects. This situation concerns 8% of our samples.

2/ The use of 6-TG as standard gives 6-TGN concentrations in the therapeutic
range while the use of 6-TGR standard gives 6-TGN concentrations above
therapeutic range. In this situation, patients exhibit high 6-TGN values with
risk of side effects. This situation concerns 21% of our samples.

These preliminary results need to be confirmed with supplementary data.


DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported
in this paper.


ACKNOWLEDGEMENTS

The authors thank Caroline Michard and Pierre Emmanuel Ubaud for their technical
assistance.

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