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Antimicrobial Agents and Chemotherapy, March 2006, p. 835-840, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.835-840.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Lack of Evidence for In Vivo Transformation of Zidovudine Triphosphate to Stavudine Triphosphate in Human Immunodeficiency Virus-Infected Patients

Margarita Meléndez,1 Raúl Blanco,2 Wilfredo Delgado,2 Rosario García,2 Jorge Santana,3 Hermes García,4 Osvaldo Rosario,1 and José F. Rodríguez2,3*

Department of Chemistry, Río Piedras Campus, University of Puerto Rico,1 Department of Biochemistry, School of Medicine, Medical Sciences Campus, University of Puerto Rico,2 AACTU-Department of Medicine, School of Medicine, Medical Sciences Campus, University of Puerto Rico,3 Puerto Rico Health Department (CLETS), San Juan, Puerto Rico4

Received 24 August 2005/ Returned for modification 29 October 2005/ Accepted 8 December 2005


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ABSTRACT
 
The in vivo and in vitro determination of significant intracellular stavudine (d4T) triphosphate (d4TTP) concentrations in human immunodeficiency virus (HIV)-infected subjects and NS-1 cells treated with zidovudine (ZDV) has recently been reported. This study was conducted to corroborate these findings with in vivo samples from HIV-infected subjects taking ZDV and in vitro CEMSS cells incubated with different ZDV concentrations. Previously, we have reported on our validated high-performance liquid chromatography coupled with tandem mass spectrometry methodology for the simultaneous determination of d4TTP, lamivudine triphosphate, and ZDV triphosphate (ZDVTP) concentrations. Using this methodology, we monitored the d4TTP concentration in more than 100 samples from HIV-infected subjects treated with d4T. In addition, we simultaneously monitored the concentrations of d4TTP and ZDVTP in more than 500 samples from HIV-infected individuals who were taking ZDV. Finally, we performed in vitro studies by incubating CEMSS cells with 10 µM, 50 µM, and 100 µM ZDV and monitored the formation of d4TTP at 24 and 48 h. We could measure d4TTP concentrations from HIV-infected individuals with a limit of quantitation (LOQ) of 2.7 fmol/106 cells (total injection, 54 fmol). In the in vivo studies, we measured the d4TTP concentrations among patients receiving d4T treatment, but the samples from patients taking ZDV did not provide d4TTP concentrations above the LOQ. Furthermore, in vitro samples did not produce any signal for d4TTP, despite the detection of substantial ZDVTP concentrations in CEMSS cells. Thus, contrary to the previous report, we found no evidence for the in vivo or in vitro transformation of ZDVTP to d4TTP in HIV-infected subjects or CEMSS cells.


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INTRODUCTION
 
Nucleoside reverse transcriptase inhibitors are an essential part of the highly active antiretroviral therapy implemented for the treatment of human immunodeficiency virus (HIV)-infected individuals (24). Two of the most widely used nucleoside reverse transcriptase inhibitors are zidovudine (ZDV) (8) and stavudine (d4T) (7). ZDV and d4T are thymidine nucleoside analogs that need to be converted into their active forms, zidovudine triphosphate (ZDVTP) and stavudine triphosphate (d4TTP), to be effective against HIV replication (6, 14). Furthermore, it has been established that ZDV and d4T use the same intracellular mechanisms for their activation processes (6, 14), and combination therapy with these two analogs is not recommended (12, 15, 16).

Various methods (indirect and direct detection of nucleotides) have been developed for the determination of intracellular ZDVTP and d4TTP in vivo. The indirect methodologies fractionate the phosphate anabolites (ZDV monophosphate, ZDV diphosphate, ZDVTP, d4T monophosphate, d4T diphosphate, and d4TTP) by the use of ion-exchange cartridges or ionic liquid chromatography, followed by dephosphorylation and quantification of the parent drug (ZDV and d4T) by radioimmunoassays or by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) (1, 16-19, 22, 26). Another approach is to combine off-line immunoaffinity extraction and HPLC-MS/MS for the determination of ZDVTP (3). Immunoaffinity was required in this method since the direct determination of ZDVTP was affected by the interference of intracellular endogenous ATP. Alternative approaches to the direct measurement of nucleotide (d4TTP) concentrations by HPLC-MS/MS have been reported (1, 19). Although both the direct and the indirect HPLC-MS/MS approaches differ mainly in the sample processing and the chromatographic conditions, their sensitivities and selectivities are comparable (1, 3, 22).

In a recent study, Becher et al. reported on the presence of d4TTP in samples from patients receiving ZDV (2). Intracellular d4TTP concentrations ranging from 3.0 to 38.5 fmol/106 cells were found in samples from patients treated with ZDV. Furthermore, in vitro experiments with NS-1 cells and peripheral blood mononuclear cells (PBMCs) cultured in the presence of ZDV produced d4TTP signals above their limit of detection. Due to the importance of these findings in the clinical setting, where cross-resistance or toxicity could be generated, we decided to corroborate their results. We monitored the d4TTP signal in patients treated with ZDV using our validated HPLC-MS/MS methodology with limits of quantitation (LOQ) of 2.7 fmol/106 cells (total injection, 54 fmol) for d4TTP (5, 9, 22). Furthermore, in vitro experiments in which ZDV (10 µM, 50 µM, and 100 µM) was incubated in CEMSS cells at 24 and 48 h were performed to corroborate the results reported previously for the conversion of ZDVTP to d4TTP in HIV-infected patients treated with ZDV.


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MATERIALS AND METHODS
 
Chemicals. d4T, azidouridine (AZdU), chloro-ATP (Cl-ATP), ammonium formate, N,N-dimethylhexylamine (DMH), formic acid, sodium acetate, and acid phosphatase (type XA) were obtained from Sigma Chemical Co. (St. Louis, MO). ZDV, ZDVTP, and d4TTP were purchased from Moravek Biochemicals (Brea, CA). Potassium chloride, acetonitrile, methanol, and glacial acetic acid (ACS certified) were obtained from Fisher Scientific (Fairlawn, NJ). Strong anion-exchange Sep-Pak plus (SAX-QMA) cartridges were purchased from Waters Co. (Milford, MA). XAD resin was obtained from Serva (Heidelberg, NY). RPMI 1640, glutamine, nonessential amino acids, penicillin-streptomycin, and fetal bovine serum were obtained from BioWhittaker (Baltimore, MD).

Standard solution preparation. ZDVTP and d4TTP standard solutions (five different concentrations) were prepared by serial dilution, starting with a stock concentration of 20 µM. AZdU was used as the internal standard.

Cell culture with ZDV and d4T. CEMSS cells were cultured with 0 µM (control), 10 µM, 50 µM, and 100 µM ZDV or d4T for 24 and 48 h; and the metabolites (ZDVTP and d4TTP) in all samples were monitored by HPLC-MS/MS.

Sample collection and processing from HIV-infected patients. Patients or their legal guardians signed an informed consent approved by the Medical Sciences Campus Institutional Review Board at the University of Puerto Rico. All patients were enrolled in intensive pharmacokinetic (PK) trials, in which the AIDS Clinical Trials Group adherence questionnaire was administered as part of the inclusion criteria and on the day before the intensive sampling for PK analysis. On the day of the PK study, directly observed treatment was supervised by one member of the clinical trial team. No food restrictions were required for the patients in the clinical trials. Blood samples (8 ml from pediatric patients or 32 ml from adult patients) were drawn and placed in cell preparation tubes with sodium heparin (Vacutainer CPT; Becton Dickinson, Franklin Lakes, NJ). Sampling time points were 0 h (predosing) and 1, 3, and 6 h postdosing for HIV type 1 (HIV-1)-infected pediatric patients treated with ZDV therapy (300 mg twice a day [BID]). The adults in the studies were sampled at 0 h (predosing) and 1, 2, 4, 8, and 12 h postdosing for those receiving the standard ZDV dose (300 mg BID) and at 0 h (predosing) and 1, 2, 4, 8, 12, 16, and 24 h postdosing for those receiving d4T (40 mg BID). PBMCs were separated from erythrocytes by centrifugation at 1,500 x g for 20 min at room temperature (10 x 106 to 40 x 106 cells). PBMCs were recovered and counted in a Coulter (Hialeah, FL) Z2 series system, followed by extraction with 70% methanol-Tris (15 mM, pH 7.4), and were stored at –80°C until analysis. All the samples were obtained and processed by the same group at the University of Puerto Rico General Clinical Research Center. The methodology for the processing of the samples has been described in the literature (5, 22, 23).

Liquid chromatography-tandem mass spectrometry. HPLC analysis was performed on an Agilent (San Fernando, CA) 1100 system with a Phenomenex Luna C18 reversed-phase column (100 mm by 2.1 mm; 3 µm). The mobile phase consisted of a methanol and acetonitrile mixture (30:10; vol/vol) with 0.25% acetic acid at a flow rate of 50 µl/min. An injection of 20 µl was sufficient for the detection of ZDV, d4T, and AZdU. A Waters (Boston, MA) Quattro II triple quadrupole mass spectrometer was used for the analysis in the multiple-reaction-monitoring (MRM) mode. Sample introduction was through an electrospray ionization source in the positive ion mode. The cone voltage was optimized for each nucleoside (and was between 10 and 15 V), and the source temperature was 120°C. Ions were activated by collision at the individual optimum energy (between 8 and 15 eV), with a cell pressure of approximately 7 x 10–4 mbar argon. MRM data were acquired in a single function with three different transitions for the parent nucleosides (ZDV, 268 to 127; d4T, 225 to 127; AZdU, 254 to 113) and were analyzed by using MassLynx software (v. 3.3).

Data analysis. Analyte concentrations were determined by using peak-area ratios for ZDV/AZdU and d4T/AZdU. Calibration curves from the ZDVTP and the d4TTP standard solutions were prepared with each batch analyzed. Linear regression analyses were performed by using five ZDVTP and d4TTP standard concentrations. Regression coefficients (r2) were better than 0.990 for all calibration curves.


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RESULTS
 
LOQs for ZDVTP and d4TTP. Calibration curves were obtained by using different standard concentrations of ZDVTP and d4TTP spiked into PBMCs and passed through the entire validated methodology. Results for the d4TTP validation method showed a coefficient of variation <11%, an accuracy of <13%, and sample recoveries above 95% in six different set of experiments (5, 9, 22). Previously, we have reported similar results for the validation of ZDVTP (coefficient of variation, <8%; accuracy, <10%; sample recovery, >95%) (5, 22). The regression coefficients for the calibration curves for both compounds were better than 0.990.

Figure 1 shows the ZDV and d4T ion chromatograms for the LOQs obtained for ZDVTP (40 fmol) and d4TTP (54 fmol) by use of our validated assays (5, 9, 22, 23). The ion chromatograms for ZDV and d4T are presented since we used an indirect method to quantify ZDVTP and d4TTP, where the phosphate groups are removed before the HPLC-MS/MS analysis. In this study, the LOQs are lower than those previously reported by our group (5, 9, 22, 23). The reason for this improvement was a modification of the method of sample introduction into the MS source, where the whole sample is introduced from the HPLC system without flow split. In addition, MRM data acquisition was modified to detect all the transitions with their respective optimum parameters as a single function instead of multiple functions, which decreased the delay between detection channels and which increased the sensitivity to the attomole range. Thus, our methodology is capable of quantifying simultaneously ZDVTP and d4TTP concentrations above 40 and 54 fmol (total injection), respectively. We determined that the ZDV signal did not interfere with the d4T signal and that the d4T signal did not interfere with the ZDV signal (data not shown). In addition, endogenous nucleotides did not interfere with the process of quantitation of ZDVTP or d4TTP, which was evident by the lack of additional signals in the ion chromatograms.


Figure 1
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  		FIG. 1. Chromatograms showing the signals for the LOQ for ZDVTP (upper panel; 40 fmol) and d4TTP (bottom panel; 54 fmol). The retention times for ZDVTP and d4TTP were 3.2 min and 2.4 min, respectively. The parent ion-to-daughter ion transitions that are monitored during the chromatographic run are for the nucleosides ZDV (268 to 127) and d4T (225 to 127) instead of the triphosphate moieties, ZDVTP (506 to 159) and d4TTP (463 to 159), since we used an indirect method to quantify the amounts of ZDVTP and d4TTP. The chromatographic conditions are described in the Materials and Methods section. The y axis is the ion intensity obtained for each compound.

Patient samples. d4TTP concentrations were determined for HIV-1-infected patients receiving 40-mg d4T therapy (BID). Figure 2 shows the d4T ion chromatogram from the d4TTP and AZdU (internal standard) ion chromatogram after 2 h postdosing. Similar results were obtained with more than 100 samples from 15 HIV-monoinfected patients accrued for an intensive PK study. We did not observe any peaks in the ion chromatogram that coeluted with the d4T signal, corroborating the fact that we did not have endogenous interference peaks. The d4TTP concentration range observed in this patient population was from 10 to 19 fmol/106 cells (Table 1). These results are consistent with the concentrations reported previously (1, 19).


Figure 2
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  		FIG. 2. Ion chromatograms for d4TTP (upper panel) and AZdU (bottom panel) from an HIV-infected patient at 2 h postdosing with d4T (40 mg BID). The parent ion-to-daughter ion transition that is monitored during the chromatographic run is for the nucleoside d4T (225 to 127) instead of the triphosphate moiety, d4TTP (463 to 159), since we used an indirect method to quantify the amount of d4TTP. The chromatographic conditions are described in the Materials and Methods section. The y axis is the ion intensity obtained for each compound.


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  		TABLE 1. Intracellular ZDVTP and d4TTP concentrations in PBMCs from patients taking either zidovudine or stavudine

ZDVTP and d4TTP concentrations were determined for HIV-1-infected pediatric patients, HIV-1-monoinfected patients, and HIV- and hepatitis C virus-coinfected patients taking ZDV (300 mg BID) (Table 1). The ZDV and d4T signals coming from their corresponding triphosphate moieties (ZDVTP and d4TTP) were monitored simultaneously by HPLC-MS/MS by using the same sample (10 to 40 x 106 cells). The ZDVTP concentration ranges were similar for the three groups (approximately 2 to 200 fmol/106 cells), with a higher mean for children (54 fmol/106 cells), since we did not monitor the concentrations in this patient population at 8 or 12 h. Figure 3 shows a 12-h pharmacokinetic profile for one HIV- and hepatitis C virus-coinfected patient receiving ZDV. The panels on the left show the signals for ZDV at the different times (0, 1, 2, 4, 8, and 12 h), while the panels on the right show the d4T ion chromatograms. The signal for ZDV is always observed above the LOQ, whereas there is no discernible signal for d4T at any of the time points. These results were similar for all the patients studied (pediatric patients, adult patients, and patients of both genders) and samples from our clinical protocol tested (>500 samples). Thus, there is no doubt that d4TTP was not observed in this time frame before administration of the next ZDV dose. This patient population had received ZDV treatment for at least 12 weeks prior to the clinical protocol, so they had an ample amount of time to produce measurable d4TTP if this in vivo process was occurring physiologically. If any d4TTP was produced in this population, the amounts were under our LOQ (<54 fmol).


Figure 3
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  		FIG. 3. Ion chromatograms for the simultaneous determination of ZDVTP (left panels) and d4TTP (right panels) obtained from an HIV-infected individual during an intensive PK sampling study (0, 1, 2, 4, 8, and 12 h postdosing). The signal for ZDVTP changes with time and is always above the LOQ, but there is no signal for d4TTP, despite the production of substantial amounts of ZDVTP. The parent ion-to-daughter ion transitions that are monitored during the chromatographic run are for the nucleosides ZDV (268 to 127) and d4T (225 to 127) instead of the triphosphate moieties, ZDVTP (506 to 159) and d4TTP (463 to 159), since we used an indirect method to quantify the amounts of ZDVTP and d4TTP. Similar results were obtained for ZDV monophosphate and d4T monophosphate. Chromatographic conditions are described in the Materials and Methods section. The y axis is the ion intensity obtained for each compound.

CEMSS cell samples. In vitro experiments were performed to determine if our in vivo observations were correct or the d4TTP concentrations in the patient population were so low that they were below our LOQ. Two different sets of experiments were performed with CEMSS cells. We cultured CEMSS cells in either the absence of ZDV or d4T (0 µM; control) or the presence of 10 µM, 50 µM, and 100 µM ZDV or d4T. The samples (40 x 106 cells) were analyzed by the methodology mentioned above, and we monitored the signal for d4T and ZDV simultaneously for both sets of experiments (Table 2). Figure 4 shows the ZDV and d4T ion chromatograms for CEMSS cell samples cultured with 100 µM ZDV or 100 µM d4T. From the ion chromatograms, the signal for ZDV coming from ZDVTP is clearly observed with a concentration of 821 fmol/106 cells (Fig. 4, upper panel), whereas no signal is present for d4TTP (Fig. 4, middle panel). However, when CEMSS cells were cultured with 100 µM d4T (Fig. 4, lower panel), a significant signal for the formation of d4TTP could be observed (1,967 fmol/106 cells). Similar results were obtained with concentrations of 10 µM and 50 µM (Table 2). Thus, we did not find any evidence for the formation of d4TTP when CEMSS cells were cultured with ZDV.


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  		TABLE 2. Intracellular ZDVTP and d4TTP concentrations in CEMSS cells when the cells were incubated in the presence of various zidovudine or stavudine concentrations


Figure 4
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  		FIG. 4. ZDVTP ion chromatogram (upper panel) obtained from CEMSS cells cultured with 100 µM ZDV for 48 h. The middle panel shows the signal obtained for d4TTP under the aforementioned conditions. The lower panel shows the signal for d4TTP from CEMSS cells cultured with 100 µM d4T for 48 h. Similar results were obtained with ZDV concentrations of 10 µM and 50 µM for 24 h and 48 h. The parent ion-to-daughter ion transitions that are monitored during the chromatographic run are for the nucleosides ZDV (268 to 127) and d4T (225 to 127) instead of the triphosphate moieties, ZDVTP (506 to 159) and d4TTP (463 to 159), since we used an indirect method to quantify the amounts of ZDVTP and d4TTP. Chromatographic conditions are described in the Materials and Methods section. The y axis is the ion intensity obtained for each compound.


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DISCUSSION
 
The results presented above are in contrast to those presented in a previous report by Becher et al., in which they showed measurable concentrations of d4TTP (3 to 38.5 fmol/106 cells) using a direct method with samples from HIV-infected patients taking ZDV (2). ZDVTP concentrations were reported in only 64% (20 of 31) of their patient samples, with a range of from 31 to 386 fmol/106 cells. The level of conversion of ZDVTP to d4TTP ranged from 3.0% to 36.7%, although the concentration in some samples could not be quantified since no ZDVTP concentration could be detected. The ZDVTP concentrations measured in our study ranged from 2.1 to 200 fmol/106 cells in 552 samples from HIV-infected children and adults. We could quantify the ZDVTP concentrations in all the patient samples because our LOQ was 40 fmol and the numbers of PBMCs recovered was, on average, 30 x 106 cells from 8 ml (children) to 32 ml (adults) of blood. To produce sufficient d4TTP amounts (above 54 fmol) from the reduction of ZDVTP in our samples, the conversion should be between 0.7% (high ZDVTP concentrations [above 150 fmol/106 cells]) and 27% (low ZDVTP concentrations [below 5.0 fmol/106 cells]). The percent conversion shown by Becher et al. (2) is higher than the one needed for our methodology for the detection of d4TTP; thus, we can be certain that the reduction is not occurring in our patient population.

Since our methodology is different from that of Becher et al. (2) (indirect versus direct), we decided to implement the direct quantification of d4TTP using DMH as the ion-pairing reagent. Handling of DMH was cumbersome because of its poor solubility, and residues were impregnated rapidly in the mass spectrometer ion source, making the cleaning procedure more recurrent and difficult. Furthermore, the stabilization of the column with the mobile phase at a pH of 11.5 was inconsistent, and it took numerous runs (more than 5; each run is 26 min) to avoid interference peaks in the chromatogram even when Milli-Q water was injected. This method was very difficult to implement, and we could not obtain reproducible results, not even with standard solutions. This inconsistency in reproducibility was also shown in the previous work of Becher et al. (1) for the quantification of deoxycytidine triphosphate (dCTP), in which the concentration of this compound could be determined directly in the presence of other exogenous and endogenous nucleotides (1). However, a more recent report indicated that it was necessary to use sodium periodate to remove ribonucleotide interference peaks for determination of the dCTP concentration (13). Furthermore, some peaks even appear near the retention time of d4TTP, although the signal in the ion chromatogram is specific for this moiety (2).

From our ion chromatograms, it was shown that no interference peaks from other antiretroviral agents or endogenous nucleotides appeared near the retention times for ZDV and d4T. This is an important finding, since in the direct determination of ZDVTP by Becher et al. (1, 3), there was a substantial interference signal due to ATP and dGTP, which have the same molecular mass as ZDVTP (507 Da). This substantial interference peak was the main reason why Becher et al. developed an alternative method by using immunoassay extraction coupled off-line with HPLC-MS/MS to quantify ZDVTP (3). However, their recovery of ZDVTP by use of this alternative approach was only 50%. Similar situations occurred for their determination of dCTP and deoxyguanosine triphosphate (dGTP), where splitting of the sample was necessary and sodium periodate was used to remove ribonucleotides (13). In the case of dGTP, they needed a special HPLC gradient to quantify this endogenous nucleotide, but two unknown large peaks were nevertheless observed prior to and after the dGTP signal (13). For d4TTP, an unidentified peak with an equal signal appeared in the ion chromatogram just prior to the d4TTP peak (1). Thus, other species are appearing as a result of the sample or the methodology used.

At present, it is difficult to explain the reason for the appearance of a d4TTP signal in the study of Becher et al. (1) with samples from patients taking ZDV. They used a validated method, and the results showed no evidence of degradation when the experiment was performed with ZDV standard solutions spiked into cell extracts. However, it is interesting that all 31 samples analyzed had d4TTP concentrations above their limit of detection, suggesting possible contamination during sample processing. Another difference in our methodology is the selection and detection of the daughter ion in the mass spectrometer, where we have a very selective means of selection of the mass of the pertinent nucleotide. Becher et al. (1) used a general daughter ion (pyrophosphate, m/z = 159) for the detection of the nucleotides. This may be one of the reasons why they observed additional peaks in the same ion chromatogram near the retention time of the antiretroviral agent of interest. Furthermore, the high temperature of the source for electrospray (400°C) is also a concern, since degradation or fragmentation of the compounds could occur.

It is well known in organic chemistry that the azide moiety is a good leaving group and that its conversion into an amino group is feasible (20). Chemical reduction of alkyl azides into amine derivates have been demonstrated by the use of ammonium formate (10, 20), which is used in the chromatographic conditions for the direct determination of nucleotides by Becher et al. (1). The chemical transformation of ZDVTP into d4TTP in the presence of glutathione or dithiothreitol (reducing agents) was reported previously (21). However, the conditions for this transformation used concentrations of the reducing agents (glutathione at 200 mM) higher than those present in the cells (glutathione cytoplasm concentration, 20 mM) (11). Furthermore, glutathione concentrations in HIV-infected patients have been shown to be lower that those in noninfected controls (4, 25). Another aspect that requires consideration in the chemical transformation of ZDVTP to d4TTP is the pH dependence of this reduction. Reardon et al. (21) showed that the chemical reduction was favorable at high pH values (pH 10), which is one of the chromatographic conditions in the methodology used by Becher et al. (1). Thus, the chemical conversion of ZDVTP first to 3'-amino-3'-deoxythymidine triphosphate and then to d4TTP could occur under the experimental conditions used in the direct determination.

The following arguments support our findings for the lack of evidence for the in vivo or in vitro conversion of ZDVTP to d4TTP: (i) the indirect methodology used is capable of simultaneously detecting ZDV and d4T in a single chromatographic run; thus, all the intracellular extracted nucleotides are processed and detected in a single analysis and any d4TTP formed from intracellular ZDVTP should be detected. (ii) The sensitivity of our methodology is comparable to that of the methodology of Becher et al. (2), which allows the detection of any d4TTP signal coming from ZDVTP; but we had even more sample (10 x 106 cells per sample versus 30 x 106 cells per sample), which allowed us to determine better this physiological transformation. (iii) The quantitation of d4TTP over a concentration range of 10 to 19 fmol/106 cells was achieved in HIV-infected patients taking stavudine (40 mg BID). (iv) In vitro experiments with 10 µM, 50 µM, and 100 µM ZDV never led to any d4TTP signal, whereas incubation with d4T showed substantial concentrations.

In summary, our in vivo and in vitro studies could not corroborate the intracellular transformation of ZDVTP into d4TTP. Our results clearly demonstrate that the LOQ for the determination of d4TTP (54 fmol) is sufficient to observe the amounts previously reported for the transformation. Neither the 552 patient samples obtained over the past year nor the CEMSS cell samples treated with ZDV showed any signal corresponding to d4TTP. Further studies are necessary to determine the reasons for the discrepancies in these results.


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ACKNOWLEDGMENTS
 
This work was supported in part by the following Public Health Service grants: 2U01AI32906, 1P20RR11126, G12-RR03051, AI34858, R25-GM61838 (to M.M.), and R01AI39191 (to J.F.R.).

We acknowledge the technical assistance of Ileana Feliciano.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, P.O. Box 365067, School of Medicine, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico 00936-5067. Phone and fax: (787) 754-4929. E-mail: jrodriguez{at}rcm.upr.edu. Back


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Antimicrobial Agents and Chemotherapy, March 2006, p. 835-840, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.835-840.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Susan-Resiga, D., Bentley, A. T., Lynx, M. D., LaClair, D. D., McKee, E. E. (2007). Zidovudine Inhibits Thymidine Phosphorylation in the Isolated Perfused Rat Heart. Antimicrob. Agents Chemother. 51: 1142-1149 [Abstract] [Full Text]  
  • Benech, H., Becher, F., Pruvost, A., Grassi, J. J., Melendez, M., Rodriguez, J. F. (2006). Is Stavudine triphosphate a natural metabolite of Zidovudine?. Antimicrob. Agents Chemother. 50: 2899-2901 [Full Text]  

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