Anal. Chem. 1998, 70, 1664-1669
Measuring DNA Synthesis Rates with [1-13C]Glycine Pei Chen and Fred P. Abramson*
Department of Pharmacology, George Washington University, School of Medicine and Health Sciences, 2300 I Street NW, Washington, D.C. 20037
We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.
We have devised a nonradioactive, nontoxic method to measure DNA synthesis rates. This fundamental measurement of cellular replication, so frequently performed in experimental systems with [3H]thymidine (3H-dT) or bromodeoxyuridine (BrdU), is not readily measured in the clinical setting. The doses of radioisotope and the subsequent incorporation into the cell nucleus inhibit its use. Although increasingly popular, halogenated pyrimidines, such as BrdU, are toxic and mutagenic. In contrast, stable isotopes as tracers have a 50+ year history of safety. Use of stable-isotope-labeled substances, e.g., glycine as a precursor of de novo purine biosynthesis, averts any concerns about safety and would encourage the clinical use of a fully 1664 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
developed method to study fundamental processes, diseases, and therapies that affect cellular proliferation. For these measurements, we have used chemical reaction interface mass spectrometry (CRIMS), which has evolved from a basic concept into a selective, sensitive, and versatile technique by which targeted isotopes or elements can be monitored in studies of metabolism.1 CRIMS parallels the use of radioisotopes in that this monitoring takes place without concern for the chemical structures in which the targeted species exist. Using CRIMS, intact analytes are first decomposed to their elemental species in a high-temperature electronic plasma and then interact with atoms of a reactant gas to form a set of new, small, polyatomic species that are detected by a mass spectrometer. The presence of a given polyatomic species signifies the presence of a particular element, the abundance of that species quantifies it, and the isotopic signature of that species can differentiate enriched from endogenous materials. Previously, we have compared how well HPLC/CRIMS can detect stable isotopes in a drug metabolism study with the standard form of measurement that uses 14C and radioactivity monitoring.2 Following administration of a mixed dose of both radioisotope and stable-isotope-labeled tirilazad, we carried out a parallel set of high-performance liquid chromatographic analyses for drug metabolites in bile samples from monkey and dog. The comprehensiveness of detection, chromatographic resolution, sensitivity, signal/noise, and quantitative abilities of CRIMS were compared with radioactivity monitoring (RAM) and in no case was RAM superior. With HPLC/CRIMS, stable isotopes such as 13C and 15N can be comprehensively detected and quantitative patterns of drug metabolism from biological fluids can be produced that mirror the results when 14C is used. Therefore, stable isotopes may be substituted for radioisotopes in studies of metabolism where the ability of the latter approach to detect a label independent of the structures in which the label appears has been the primary reason for continuing to use a hazardous tracer. Beyond the capabilities of conventional mass spectrometers is a special configuration of a mass spectrometer optimized for making stable-isotope abundance measurements, the multicollector isotope ratio mass spectrometer (IRMS). An IRMS is designed to accept gaseous samples, such as CO2, ionize this gas with high efficiency, and transmit the appropriate ions to a multiple collector (1) Abramson, F. P. Mass Spectrom. Rev. 1994, 13, 341-356. (2) Abramson, F. P.; Teffera, Y.; Kusmierz, J.; Steenwyk, R. C.; Pearson, P. G. Drug Metab. Dispos. 1996, 24, 697-701. S0003-2700(97)01294-8 CCC: $15.00
© 1998 American Chemical Society Published on Web 03/27/1998
described, it is important that dT is not labeled by the tracer glycine molecule, because the 13C isotope ratio of dT will serve as a control for any enrichment found in deoxyguanosine or deoxyadenosine. Therefore, our first experiments used [1-13C]glycine as the purine precursor. We have begun our studies with HEP G2 human hepatoma cells. This standard cell line was selected because of its reasonable growth conditions and current availability in the department’s cell culture facility. HEP G2 cells are frequently used for metabolic studies, in particular studies of lipids and lipoprotein synthesis.9 They grow well and to good densities, thus making them practical to use. Figure 1. Origin of carbon and nitrogen atoms in purines (after Lehninger8).
that monitors two or three ions at the same time. The use of IRMS allows measurements of natural variations in the abundance of 13C and 15N3 and extends the range of tracer incorporation orders of magnitude below what is accomplished with a conventional GC/MS or HPLC/MS system.4,5 Before CRIMS, coupling chromatography to an IRMS involved a combustion interface.3 We have joined chromatography with IRMS using our chemical reaction interface.6 The most important aspect of this development was to construct a continuous-flow HPLC/IRMS combination. With our system, the precision of isotope ratio measurements with HPLC introduction is equal to the same system with GC introduction.6 Tritiated thymidine is the standard method for measuring DNA synthesis rates. Specific effects of agents that stimulate cell proliferation or slow the progression through the cell cycle can be quantified. The halogenated pyrimidine techniques developed for flow cytometry and microscopy have almost replaced autoradiographic techniques with 3H-dT in cell kinetic studies. The most widely used is the BrdU technique, which is sensitive, fast, and easy to carry out. The essence of the procedure is to pulse-label cells with BrdU by a short incubation in vitro or by a single injection in vivo. Then the cells are stained using a monoclonal antibody against BrdU and analyzed using a flow cytometer or microscope. In cell cultures, continuous exposure to BrdU may entail a plethora of adverse effects on cellular functions, including metabolic changes due to alterations in the balance of nucleotide pools, direct DNA damage, and alterations in DNA-protein interaction.7 The de novo biosynthesis of nucleotides is well described.8 Both carbons and the nitrogen of glycine are incorporated into purines (see Figure 1). A possible problem here is that C2 of glycine is directly converted into a tetrahydrofolate derivative and intermixes with C2 and C3 of serine. One of those serine carbons might also subsequently become the one carbon group in folate. Folate is involved in de novo thymidine biosynthesis. As will be (3) (4) (5) (6)
Brand, W. A. J. Mass Spectrom. 1996, 31, 225-235. Goodman, K. J.; Brenna, J. T. Anal. Chem. 1992, 64, 1088-1095. Bier, D. M. Eur. J. Pediatr. 1997, 156 (Suppl. 1), S2-S8. Teffera, Y.; Kusmierz, J. J.; Abramson, F. P. Anal. Chem. 1996, 68, 18881894. (7) Ormerod, M. G. Flow Cytometry: A Practical Approach, 2nd ed.; Oxford University Press Inc.: New York, 1994; p 165. (8) Lehninger, A. L. Biochemistry, 2nd ed.; Worth Publishers: New York, 1978; Chapter 26.
EXPERIMENTAL SECTION Cell Culture. HEP G2 cells were grown in 25 mL of supplemented phenol red-free Dulbecco’s minimal essential media (Sigma Chemical Co., St. Louis, MO) by plating (2.5-10) × 106 cells into 150-cm2 culture flasks. Each liter of the media was supplemented with 10% fetal calf serum, 1% penstrep, 4.5 g of glucose, 2.7 g of sodium bicarbonate, 0.58 g of glutamine, and 0.11 g of pyruvate. The final pH was adjusted to 7.1-7.3. Fetal calf serum and penstrep were obtained from Biofluids (Rockville, MD). All other chemicals were cell culture grade from Sigma. This medium contains 0.4 mM glycine. With 13C labeling, various amounts of [1-13C]glycine (99%, Cambridge Isotope Laboratories, Andover, MA) were added to achieve the desired level of enrichment. The cells were cultured at 37 °C, with 95% relative humidity and a CO2 level of 5%. The cells were fed every 2 days with 25 mL of media and were split 1 to 10 every week. DNA Isolation. DNA was extracted by using the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN). All enzymes used were obtained from Boehringer Mannheim (Indianapolis, IN). Two or three flasks containing a total of (1-2) × 108 cells are harvested on desired days and washed with PBS solution. The cells are transferred to a 50-mL tube and centrifuged at 500g for 5 min. The supernatant is removed, and 30 mL of cell lysis buffer is added. Next, proteinase K is added to a final concentration of 100 µg/mL and incubated 2 h at 37 °C. We next add RNase at a final concentration of 20 µg/mL and incubate for 2 h at 37 °C. Then we add 10 mL of the Puregene’s protein precipitation solution to the lysed cells, vortex for 30 s, and centrifuge at 2000g for 10 min. The supernatant containing the DNA is poured into a new tube containing 25 mL of iPrOH (leaving behind the protein pellet), and we mix the sample by inverting gently 50 times until the white thread of DNA is visible and again centrifuge at 2000g for 10 min. DNA will form a white pellet. We pour off the supernatant and drain the tube on filter paper. Then we add 30 mL of 70% EtOH, invert the tube several times to wash the DNA, centrifuge at 2000g for 5 min, and carefully pour off the EtOH. We drain the tube on filter paper and allow the sample to air-dry for 5 min. Finally, we add 2.5 mL of H2O and rehydrate the DNA overnight (or heat at 65 °C for 1 h). Digestion of DNA. DNA samples were enzymatically hydrolyzed to nucleosides using a modified procedure described by Saris et al.10 All enzymes used were also from Boehringer Mannheim. The DNA is denatured by heating in boiling water (9) Javitt, N. B FASEB J. 1990 4, 161-168. (10) Saris, C. P.; Damman, S. J.; van den Ende, A. M. C.; Westra, J. G.; den Engels, L. A. Carcinogenesis 1995, 16, 1543-1548.
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Figure 2. HPLC/CRI/IRMS chromatogram of DNA nucleosides extracted from HEP G2 cells. The peak eluting at 11.2 min is unknown. The amplifier for m/z 45 has a 100× gain compared with m/z 44.
for 3 min and then chilled rapidly with ice water. To a solution of denatured DNA (0.5 mg/mL), the following were added (per mL): 100 µL of 10× buffer (20 mM MgCl2, 10 mM ZnCl2, 500 mM Tris, pH 7.2); 10 µL of DNase I (0.5 unit/µL); 10 µL of Nuclease P1 (0.5 unit/µL); and 20 µL (4 munits/µL) of phosphodiesterase. The solution was incubated for 2 h at 37 °C. Then 1 µL of 10 M ammonium acetate (pH 9.0) and 5 µL of alkaline phosphatase (1 unit/µL) were added and incubated for another 2 h at 37 °C. Nucleoside Purification and Analysis. HPLC solvents were obtained from EM Science (Gibbstown, NJ) with less than 0.1 ppm evaporation residue. All HPLC experiments were conducted with a pair of Isco model 260D syringe pumps (Isco Inc., Lincoln, NE) coupled with a Gilson model 811C dynamic mixer (Gilson Co. Inc., Middleton, WI). Samples were dissolved in deionized water and injected using a Model 7125 valve (Rheodyne, Coati, CA) with a 100-µL loop. The nucleoside mixture was filtered with a 0.22-µm nylon filter (Micron Separations Inc., Westboro, MA) and purified using a Waters (Milford, MA) Nova-Pak C18 column (8 × 100 mm, 60 Å). The mobile phase A was 5 mM ammonium acetate (pH 4.0) and mobile phase B was 50:50 acetonitrile/water (v/v). The solvent conditions were 0% B for 5 min and then a linear gradient to 90% B in 5 min. The flow rate was 2 mL/min. The nucleoside portion was collected and concentrated using a Speed Vac. The nucleosides were injected into a 100-µL loop and separated using a Supelco (Bellefonte, PA) 18-S column (4.6 × 250 mm, 5 µm) before entering the CRI/IRMS. The gradient used was from 5% to 20% B in 15 min (linear) at 1 mL/min. As seen in the accompanying chromatogram (Figure 2), excellent separations were obtained with this procedure. Part of the purification removes a major impurity, but that also mostly removes deoxycytidine (dC). dT is unaffected by the purification so that dT becomes the preferred internal standard for isotope ratios when glycine is the labeled test substance. The isotopic analysis was carried out using a Finnigan/MAT Delta S IRMS (Finnigan/MAT, San Jose, CA) to which we had added our HPLC/CRI interface.6 The CRI reactant gas was UHP oxygen (Matheson Gas Products, East Rutherford, NJ). The integration of each peak used a slope sensitivity of 1 mV/s, a value that we found gave the most 1666 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
reproducible values. Most samples were analyzed three to five times and the standard deviations plotted. Measurement and Kinetic Analysis of Growth and Decay. A preliminary set of experiments was carried out where 33% enrichment with [1-13C]glycine was used. The isotope ratio of an internal standard, 5′-fluoro-2′-deoxyuridine (Sigma), was determined independently (δ13C ) -25.3‰11) and initially δ13C values for the nucleosides were based on that isotope ratio. From the results of this preliminary experiment, we defined a lower degree of enrichment for our subsequent work. The second study followed growth of label over 5 days during continuous incubation with glycine enriched to 3.3%. In the third study, different amounts of HEP G2 cells were first incubated with a 1-day pulse of 9% enriched glycine and then the medium was exchanged for unlabeled medium. We measured the washout of the incorporated label from these pulsed cells over the next 9 days. Because we need the same number of cells to make our measurements, we started with relatively more cells for analysis on days 0 and 1 than on the later days. The cells that grew for 7 and 9 days were split once to maintain their log growth characteristics. The labeling rate for cells in log-phase growth will follow a first-order kinetic model. Therefore, the data from these experiments were fit with the suitable exponential growth or decay equation. For incorporation of label we used
δ13Ct ) δ13Css(1 - e-kt) and for wash out of label we used
δ13Ct ) δ13C0e-kt The curve-fitting procedure in SlideWrite Plus for Windows, Version 4.0 (Advanced Graphics Software, Inc., Carlsbad, CA) was used to obtain the parameters of these equations and the correlation coefficients of the data to the appropriate equation. The rate constant, k, was converted into a doubling time using ln(2)/k. One of the two methods we used to generate “conventional” data regarding the growth rate of HEP G2 cells was to use tritiated thymidine. The medium of HEP G2 cells was supplemented with 0.1 µCi/mL 3H-dT (Amersham Life Science, Arlington Heights, IL) and 1 µM unlabeled thymidine. After 1 day, some cells were transferred into medium that did not contain 3H-dT. At various time points during the incubation, the medium was removed, the adherent cells were washed three times with PBS, and the cells were scraped into 1 mL of ice cold PBS. The cell suspension was treated with 1.2 N PCA, and the precipitate was collected, washed three times with 0.2 N PCA, and solubilized in 1 N NaOH (0.5 mL). An aliquot was counted in acidified scintillation medium. These data were also analyzed with exponential growth or decay equations as was done for the labeled glycine experiments. The second method to generate comparable data regarding the rates of cell growth was to do a series of cell counts during a normal incubation of these cells. After trypsinization, the cells (11) High-precision IRMS data are conventionally expressed per mil (‰) rather than percent. The data are expressed as δ13C‰ computed as 1000(IRX IRPDB)/IRPDB where PDB is an international standard isotope ratio (IRPDB) ) 0.011 237 2.
Figure 3. Growth of 13C in dA and dG in the DNA from HEP G2 cells during continuous isotopic labeling with [1-13C]glycine. No data for dG on day 5 were obtained. The fit is to the equation for exponential growth. The correlation coefficient was 0.994 for dA and 0.986 for dG.
Figure 4. Washout of 13C in dA and dG in the DNA from HEP G2 cells following a 1-day pulse of labeled glycine. The data were fit using the equation for exponential decay. The points for day 0 were not used for the fit. The correlation coefficient was 0.974 for dA and 0.994 for dG.
were pelleted and resuspended to give an approximately 200 000 cell/mL concentration. A 100-µL aliquot of that suspension was diluted in 9.9 mL of Isoton II (Coulter Corp., Miami FL). The cells were counted using a Coulter counter ZM. RESULTS AND DISCUSSION In the preliminary studies with 33% labeled glycine, deoxyadenosine (dA) enrichment rose to a δ13C of +375‰ and deoxyguanosine (dG) rose to +321‰ after 4 days of incubation. From the growth experiment with 3.3% [1-13C]glycine (Figure 3), we measured the doubling time for the HEP G2 cells to be 2.3 ( 0.4 days using the kinetics of dA enrichment and 2.2 ( 1.2 days using dG kinetics. The lesser precision of the dG component of the growth experiment is due to our inability to obtain a measurement for dG on day 5; thus the fit involves one less data point. The errors reported are the SEs for the fitted parameters. The projected steady-state enrichments were +66‰ and +63‰, respectively. In addition, we measured the isotope ratio of dT. Over these 5 days, its δ13C value was constant at -15.8 ( 1.2‰ (mean ( SD, N ) 7) compared to the value on day -1 of -15.9 ( 2.2‰ (N ) 6). This observation validated our hypothesis that [1-13C]glycine is selectively incorporated into purines and not into pyrimidines. Consequently, we felt justified to use the measured control isotope ratio for dT to calculate dG and dA enrichment at any time in a given experiment. From the washout experiment (Figure 4), we determined doubling times of 2.6 ( 0.3 days for dA and 2.4 ( 0.1 days for dG. We did not use the day 0 data because the fit was considerably better without it. The assumption that all the unincorporated label was fully and instantly washed out does not seem to be valid. The experiments using 3H-dT also were well described by these equations (Figure 5). The half-lives for growth and decay were 1.9 ( 0.2 and 1.23 ( 0.04 days, respectively. From the loglinear portion of the cell growth curve (Figure 6), the doubling time was estimated as 1.7 ( 0.2 (SE) days. Beyond showing the feasibility of this stable-isotopic method to observe the enrichment of nucleosides from de novo biosynthesis, the more important goal is to evaluate the validity of this approach. The most important observation was that, whether
Figure 5. Growth (A) and washout (B) of [3H]thymidine incorporation into DNA in HEP G2 cells grown in culture. The lines are fits to exponential growth and decay. The regression coefficient was 0.997 for growth and 0.998 for decay.
observing incorporation of label or the washout of label, and whether dA or dG was monitored, the quality of fits to the firstorder equations used were excellent. The correlation coefficients ranged from 0.974 to 0.994. In addition, the measured doubling times from the growth and washout components for both dA and dG were consistent with each other. A more sensitive measure about the goodness of fit of a model to the data is the relative standard error of the doubling time estimated from the fit. Except for the growth experiment where dG was quantified, the [1-13C]glycine experiments showed relative standard errors between 4% and 17%. The relative SE expresses the tightness of the data to the fitspoorer data yield poorer precision. In the dG growth Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
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Figure 6. Growth curve of HEP G2 cells in culture. The fit to log growth between days 2 and 12 is also shown. The correlation coefficient was 0.958.
experiment, not obtaining a usable value for day 5 meant that a smaller data set was matched up against the equation and the relative error was severalfold higher. For the experiments where 3H-dT was used to label DNA, the relative SEs were 12% and 3.2% using the same fitting procedure. We believe that this group of comparisons clearly establishes the validity of the [1-13C]glycine method. The experiment where the cells were counted gave a value for doubling time that is consistent with the expected doubling time for HEP G2 cells. Using labels, our measured doubling times varied from 1.23 to 2.6 days. We did not evaluate what the interexperimental errors in doubling times in these cells were, but substantial variability in the growth of cells from batch to batch and month to month is not unexpected. The observed range of doubling times is not critical because the goal of these experiments was not to determine the doubling time of HEP G2 cells. We wanted to evaluate whether the concept of doubling times was incorporated in the de novo stable-isotopic incorporation and washout data. The quality of the fits to exponential growth and decay, and the relative standard errors of the exponential rate constant, support our interpretation. One especially important component of the [1-13C]glycine method is confirming that dT maintains its natural abundance of 13C in the HEP G2 cells while dA and dG show enrichment. In living systems, it is known that δ13C values differ, depending on diet or environment,12 and such variations make observing small changes difficult because a baseline needs to be established for each subject. Ultimately, the use of dT as an internal isotopic control against which dA and dG enrichment are measured will need to be further validated by examining the dT vs dA and dG isotope ratios in control subjects. As Figure 1 shows, the carbons in purines come from a variety of sources. The de novo biosynthesis of pyrimidines involves different carbon sources, aspartate and CO2 with the thymidine methyl group coming from N5,N10-methylene-tetrahydrofolate.8 Further investigation in vitro and in experimental animals will lead to a yet better defined and validated stable-isotopic method for measuring DNA synthesis rates in human subjects. A substantial volume of literature supports the ultimate use of the work we present here. A number of experiments that are conceptually similar to our proposed technique of glycine incor(12) Kennedy, B. V.; Krouse, H. R. Can. J. Physiol. Pharmacol. 1990, 68, 960972.
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poration into purines have been done. In mice, Zaharevitz et al.13 were able to trace the incorporation of a stable-isotopic label from alanine into the pyrimidine, uracil. The incorporation of 15N in uracil from liver and intestine was linear with time. Strong et al.14 grew L1210 leukemia in mice housed in an enriched 13CO2 environment. They found a 22% increase in the 13C/12C ratio in uridine in tumors. Drugs that inhibit de novo synthesis, such as 6-azauridine, dropped this enrichment to 1.4%. These two experiments show that in vivo approaches using stable isotopes are feasible, and the de novo biosynthesis rates as measured by isotopic incorporation can be affected by suitable drugs. Still more work supports our experimental strategy. Berthold et al.15 examined hepatic RNA, not DNA, synthesis by feeding poultry and mice with algae that had been highly enriched with 13C. They found enriched intact pyrimidines, but not fully enriched purines, implying that purines are not salvaged to nearly the same extent as pyrimidines. Purines were enriched at the M + 2 position, confirming the ability of 13C2-labeled glycine to form purines by de novo biosynthesis. Although an amino acid precursor was not used, an important precedent for our ultimate type of application comes from Heck et al.16 They gave five ip doses of both stable (500 ng/g per dose) and radioactive-labeled forms of thymidine to a rat and measured the incorporation of both types of isotopes into DNA from small intestine, bone marrow, spleen, thymus, liver, and brain. The sensitivity of their method of stable-isotope analysis, field ionization mass spectrometry, is difficult to evaluate today because this technique is no longer in favor and they did not provide any specific information regarding their minimum detection limits. The fact that they were able to quantify enrichment shows that this type of experiment can be done in vivo. Furthermore, they found parallel values for these two types of measurements, supporting the idea that a stable-isotope-labeled material could be a suitable analogue for the standard 3H-dT procedure. Their stable-labeled thymidine was detected at M + 6, where there was no interference from the unenriched thymidine. With [1-13C]glycine, only one atom of 13C is introduced into a purine, and conventional mass spectrometric methods would have to monitor for enrichment at M + 1, where the natural abundance of isotopes would greatly limit the detection. Only with an isotope ratio monitoring method that reformulates the analyte into CO2 can low enrichments of a single isotopic substitution be determined. HPLC/CRI/IRMS appears ideal for this purpose. Most recently, Macallan et al.17 described labeling the deoxyribose moiety in DNA by using [6,6-2H2]glucose in cell culture, rats, and humans. After hydrolysis, they used trimethylsilylation and conventional GC/MS detection to measure enrichment in dA. In HEP G2 cells, a high correlation was obtained between dA enrichment and direct cell counts. Approximately 500 mg was infused into rats and 60 g was given to each human subject. Rates (13) Zaharevitz, D. W.; Anderson, L. W.; Strong, J. M.; Hyman, R.; Cysyk, R. L. Eur. J. Biochem. 1990, 187, 437-440. (14) Strong, J. M.; Anderson, L. W.; Monks, A.; Chisena, C. A.; Cysyk, R. L. Anal. Biochem. 1983, 132, 243-253. (15) Berthold H. K.; Crain, P. F.; Gouni, I.; Reeds, P. J.; Klein, P. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10123-10127. (16) Heck, H. d’A.; McReynolds, J. H.; Anbar, M. Cell Tissue Kinet. 1977, 10, 111-119. (17) Macallen D. C.; Fullerton C. A.; Neese R.; Haddock K.; Park S.; Hellerstein M. K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 708-713.
of cellular proliferation in intestine, liver, and thymus were obtained from the rats, and granulocyte kinetics were monitored from the patients. These authors state, as do we, that a validated, nontoxic, nonradioactive method for measuring DNA synthesis rates in vivo has many important biological applications. There are a number of ways that the assay of Macallan et al.16 can be compared to the present method. Their use of labeled glucose does not permit any nucleoside to serve as an internal standard because all deoxyriboses will be enriched while the selectivity of [1-13C]glycine labeling preserves this feature. The conventional GC/MS analysis involving silylation used by Macallan et al. means that the limits of detection for enrichment will be poor. From the isotopic contributions of the three Si atoms, as well as the C, H, O, and N it contains, we calculate that the M + 2 mass at which they measure enrichment has 16% abundance relative to the unenriched mass. As compared to an isotope ratio monitoring approach that measures CO2 with its 1% natural abundance, this GC/MS procedure is working with a 16-fold higher background. In culture, glucose appears to label DNA more efficiently than glycine. With 10-15% enrichment of glucose, the lowest enrichment from HEP G2 cells reported by Macallan et al.16 was 6% for dA at 36 h. With 3.3% [1-13C]glycine, our enrichment at 36 h is estimated to be 0.03% (a ∆δ of 30‰; see Figure 3). Still, this is a substantial enrichment relative to the precision of the measurement, which is 1‰-2‰. However, the concentration of glucose in cell culture medium is 100-200 times higher than the concentration of glycine. Whether one uses a more or less efficient substrate to label the DNA, an isotope ratio mass spectrometer capable of detecting orders of magnitude lower enrichment5 would greatly reduce the amount of label used, which in the human studies cost $4500.18 Use of HPLC introduction avoids the substantial numbers of species added that dilute the
presence of the tracer species when the analyte is derivatized. Despite the higher efficiency of glucose to label nucleosides, between the ability of the HPLC/CRI/IRMS to measure lower enrichments and the lower concentration of the substrate we used to monitor de novo biosynthesis, our experimental approach can work with 1 order of magnitude or less labeled material than required by Macallan et al.16 There are still many aspects of the use of stable-isotopic methods for DNA synthesis to work out before its routine use in humans. In particular, what dosing schedule is optimal and what population of cells will one use for this measurement? Macallan et al.16 examined granulocytes, which are readily available from a venous blood sample. We will evaluate these questions with animal experiments. We believe that the availability of a safe, practical method for measuring DNA synthesis rates could become an important diagnostic or prognostic tool in hematology, oncology, transplantation biology, and aging, to name just a few clinical areas. The work presented here is our first step toward that goal.
(18) Cambridge Isotope Laboratories, Inc. Stable Isotopes Catalog, 1997-1998.
AC971294I
ACKNOWLEDGMENT We thank Dr. Blaire Osborn for carrying out the cell count experiment. We were assisted in the cell culture work by Dr. Katherine Kennedy, Dr. Joanne Kelleher, and Tayseer Aldaghlas. This work was supported by NSF Grant BIR9216935 and USPHS Grant NIH R01-GM36143. The purchase of the isotope ratio mass spectrometer was also supported by USPHS Grant NIH R01GM49321. A preliminary report on this work was presented at the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, June 1-5, 1996. Received for review November 26, 1997. Accepted March 9, 1998.
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