The new approach, described in Frontiers in Analytical Science by Rong Fu, Diane J. Sutcliffe, Ashok R. Dinasarapu, Ellen J. Hess and H. A. Jinnah, relies on ultra performance liquid chromatography coupled with photodiode array ultraviolet detection, commonly abbreviated UPLC-PDA. Unlike mass spectrometry-based metabolomics, which can be expensive and technically demanding, PDA detection exploits the strong ultraviolet absorbance of purines at 254 nanometers, allowing straightforward identification by retention time and spectral profile. The system consists of a Waters ACQUITY H-Class plus core with a quaternary solvent manager and an eLambda PDA detector, operating on a 2.1 by 100 millimeter reverse-phase C18 column packed with 1.8 micrometer particles at 40 degrees Celsius.

Separation chemistry is the crux of the method. Purines carry ionizable groups whose charge states shift dramatically with pH, so the Emory team designed a stepped gradient between two carefully formulated buffers. Buffer A contains 10 millimolar ammonium acetate and 1.5 millimolar tetrabutylammonium phosphate at pH 5.0, while buffer B contains 10 millimolar ammonium phosphate with the same ion-pairing reagent and 25 percent acetonitrile at pH 7.0. The tetrabutylammonium phosphate acts as an ion-pairing agent, taming the highly polar nucleotides so they interact with the reverse-phase column and elute as discrete peaks rather than co-eluting in the void volume. The gradient holds 100 percent buffer A for 11 minutes, ramps to 75 percent buffer B, then climbs to 100 percent buffer B before the column re-equilibrates for 9.5 minutes ahead of the next injection.

With this configuration, the method resolves all 15 target metabolites: ATP, ADP, AMP, adenosine, adenine, GTP, GDP, GMP, IMP, ZMP, guanosine, hypoxanthine, inosine, xanthine and uric acid. Only 5 microliters of sample are injected per run. Calibration with purified standards confirmed linear detection across a remarkable four-order-of-magnitude range from 0.1 to 100 micromolar, with all metabolites detectable down to approximately 100 nanomolar. Reproducibility proved equally impressive. When the same sample was analyzed five times within a single batch and across three batches on different days, the within-run coefficient of variance averaged 0.8 percent and the between-run variance averaged 1 percent for most analytes, comfortably under the 2 percent threshold the authors cite as evidence of excellent reliability.

The improvements over the team’s earlier HPLC-PDA method are substantial. The original technique required 65 minutes per sample; the new UPLC version cuts that to 33 minutes. Sensitivity is higher with half the sample volume, a critical advantage when working with scarce clinical specimens or small cell cultures. Perhaps most importantly, the new method cleanly discriminates GMP from IMP, two metabolites that co-eluted and could not be separately quantified with the older protocol. Because GMP and IMP occupy distinct positions in purine interconversion pathways, their separate measurement opens new analytical possibilities for studies of guanine nucleotide synthesis and purine salvage.

To demonstrate performance in genuine biological material, the researchers applied the method to induced pluripotent stem cells derived from three publicly available lines: KOLF2.0, NCRM1 and PGP-1. Each line was studied in matched pairs, with and without a c.508C>T stop codon mutation in the HPRT1 gene. This mutation abolishes hypoxanthine-guanine phosphoribosyltransferase, the salvage enzyme whose complete loss causes Lesch-Nyhan disease, a rare X-linked disorder marked by self-injurious behavior, dystonia and severe motor impairment. Cells were grown in six-well plates to roughly one million cells per well, with culture medium freshly replaced 24 hours before collection.

Sample preparation demanded meticulous attention to chemistry. For intracellular purines, cells were released by trypsinization, pelleted, and extracted in 0.2 molar perchloric acid, which instantly denatures the enzymes that would otherwise degrade labile nucleotides. Sonication at 4 degrees Celsius liberated the contents, and after centrifugation the acidic supernatant was neutralized with potassium carbonate, precipitating potassium perchlorate before final filtration through PVDF microcentrifuge tubes. For extracellular purines, 450 microliters of conditioned medium were acidified with perchloric acid to 0.1 molar and processed identically. Intracellular results were normalized to total cellular protein measured with a BCA assay, while extracellular results were corrected for culture volume and duration.

The biological findings were striking. In keeping with decades of prior work, the adenine derivatives dominated the intracellular purine pool, with ATP present in the greatest amounts, roughly 33 to 46 nanomoles per milligram protein across the lines, followed by GTP at 11 to 16 nanomoles per milligram. The adenylate energy charge, calculated as ([ATP] + 0.5 × [ADP]) / ([ATP] + [ADP] + [AMP]), ranged from 0.81 to 0.88, confirming that all cultures retained a healthy nucleotide energy state above the 0.80 benchmark typical of viable cells. Multivariate analysis of variance revealed significant main effects for both mutation status and cell type, along with a significant interaction between the two variables.

Most intracellular purines showed small or no changes in the HPRT1-mutant cells, but the extracellular compartment told a very different story. Conditioned medium contained no detectable purines except hypoxanthine, which was elevated by an average of 30-fold in the mutant lines, with individual increases ranging from 12-fold to as much as 225-fold depending on the genetic background. The explanation is elegant: without functional HPRT, the cells cannot recycle hypoxanthine back into nucleotides, so they simply dump it into the extracellular space. This finding underscores why measuring extracellular purines is essential, since changes outside the cell were far more obvious than those within it.

The authors acknowledge limitations. The method focuses on free purines and does not capture purines incorporated into DNA, RNA, NAD(H), NADP(H), FAD(H) or other complex molecules, though it can be adapted for some of these derivatives. Cyclic nucleotides and deoxynucleotides occur at levels too low for direct detection without prior sample concentration. The team also emphasizes the extraordinary speed of purine degradation: within seconds, ATP is dephosphorylated to ADP and then AMP, and within minutes AMP is further catabolized to adenosine, inosine, hypoxanthine and uric acid. Tissue measurements demand freeze-clamping in situ or focused microwave irradiation to halt degradation. Finally, the authors caution that isolated ATP measurements can be misleading, since they cannot distinguish energy impairment from wholesale purine depletion; only the simultaneous measurement of ATP, ADP and AMP, captured by the adenylate energy charge, provides a faithful picture of cellular energy state. With its speed, sensitivity and comprehensive coverage, the new UPLC-PDA method appears well suited to simultaneous analysis of the most abundant biologically relevant purines across blood, urine, cerebrospinal fluid and cultured cells, offering a powerful new lens on disorders of purine metabolism.

Subject of Research: Biomedical Analysis and Diagnostics

Article Title: Comprehensive measurement of purines in biological samples

Article References: Fu, R., Sutcliffe, D. J., Dinasarapu, A. R., Hess, E. J., & Jinnah, H. A. (2025). Comprehensive measurement of purines in biological samples. Frontiers in Analytical Science, 5, Article 1600781. https://doi.org/10.3389/frans.2025.1600781

Image Credits: AI Generated

DOI: 10.3389/frans.2025.1600781

Keywords: purine metabolism, UPLC-PDA, Lesch-Nyhan disease, hypoxanthine, adenylate energy charge, metabolomics, HPRT1, analytical chemistry, Comprehensive, measurement, purines, biological

The study focused on Madin-Darby Bovine Kidney cells, a widely used laboratory model of renal epithelial tissue. The choice of a bovine kidney line is not incidental. Livestock are chronically exposed to phthalates through contaminated feed, water, and environmental matrices, and renal epithelial cells represent a plausible first line of contact for circulating plasticizer metabolites. What the researchers found was a stark, concentration-dependent pattern of cellular injury that unfolded across multiple independent assays, each interrogating a different layer of genomic integrity.

The first layer was simple survival. Using the MTT assay, a colorimetric test in which metabolically active cells convert a yellow tetrazolium compound into purple formazan crystals, the team exposed MDBK cells to DBP concentrations ranging from 5 to 200 micromolar. Cell viability declined in a dose-dependent fashion, and the concentration that killed half the cells, the LC50, came out at 50 micromolar after 24 hours of exposure. That figure anchored the rest of the experimental design: the researchers selected 25, 50, and 75 micromolar as their working concentrations, spanning a range from sublethal stress to severe toxicity, to probe what happens to cells that survive a phthalate encounter.

What happens, it turns out, is a cascade of genetic damage. The alkaline comet assay, a single-cell gel electrophoresis technique that untangles DNA and allows broken strands to migrate out of the cell nucleus like the tail of a comet, revealed significant, dose-dependent increases in the DNA damage index and in the percentage of tail DNA. In parallel, the genomic instability index climbed with rising DBP concentration. These are not subtle statistical whispers; the damage signals rose consistently across the concentration range, and at the highest dose of 75 micromolar, the genotoxic response was comparable to that of the positive control group, the benchmark of maximal damage against which test chemicals are judged.

Chromosomal-level injury told the same story. In the cytokinesis-block micronucleus assay, cells are trapped at the point of division with cytochalasin B, and any chromosome fragments or whole chromosomes left behind during mitosis appear as micronuclei, tiny satellite nuclei outside the main nucleus. DBP-treated cells showed significant increases in micronucleus frequency and in the proportion of binucleated cells, alongside a rise in cytostasis, the halting of cell division. Meanwhile, the cytokinesis-block proliferation index, a measure of how many rounds of nuclear division cells manage to complete, fell as DBP concentration rose. Together, these results indicate that the chemical does not merely nick DNA; it leaves behind chromosomal debris that cells carry into division while simultaneously slowing the very process of proliferation.

Perhaps the most intriguing finding lies in the transcriptional response. Quantitative real-time PCR revealed that two DNA damage-associated genes, OGG1 and HPRT1, were both significantly upregulated in exposed cells. OGG1, which encodes a glycosylase that excises oxidized guanine bases, one of the most common lesions produced by oxidative stress, was elevated 5.33-fold. HPRT1, involved in the salvage pathway of purine nucleotide synthesis and long used as a sentinel of genotoxic stress, rose 6.57-fold. Crucially, higher DBP concentrations drove greater gene expression, suggesting a graded molecular alarm that tracks the dose of exposure.

The upregulation of repair genes is a double-edged signal. On one hand, it demonstrates that the cells recognized the damage and attempted to mount a repair response, a hallmark of activated DNA damage signaling. On the other hand, sustained elevation of repair machinery is itself an indicator of overwhelming or persistent genotoxic stress, and the concurrent rise in micronuclei and cytostasis suggests that the repair effort was not fully keeping pace with the injury. The authors interpret this coordinated pattern as evidence that DBP induces cellular stress and DNA damage while altering the expression of the genes tasked with fixing it, a combination that could erode genomic stability over time.

The broader context makes these results resonate. DBP is frequently detected not only in PVC consumer products but also in environmental matrices, meaning that humans, wildlife, and livestock encounter it continuously through ingestion, inhalation, and dermal contact. As an endocrine-disrupting chemical, it has already been implicated in reproductive and developmental toxicity, and regulatory frameworks in several jurisdictions have restricted its use in toys, cosmetics, and food-contact materials. What this study adds is mechanistic evidence from a renal epithelial model that DBP can act directly on the genome, independent of its hormonal effects, and that the damage is accompanied by transcriptional perturbation of the repair apparatus itself.

There are, of course, important caveats in translating a cell-culture experiment to whole organisms. The concentrations used here, particularly the 50 micromolar LC50, reflect acute exposure in vitro, and the actual internal doses experienced by animals or humans depend on absorption, metabolism, and excretion, processes that the cell line does not capture. MDBK cells, while a robust epithelial model, are bovine rather than human, and species differences in phthalate metabolism could shift the toxic threshold. Nonetheless, the internal consistency of the data, with cytotoxicity, comet-based strand breakage, micronucleus formation, proliferation arrest, and gene expression changes all pointing in the same direction across a coherent dose range, lends the findings considerable weight.

The authors conclude that DBP induces concentration-dependent cyto-genotoxicity and transcriptional alterations in MDBK cells, and they frame the result as an argument for developing safer plasticizer alternatives. As phthalates continue to leach from the built environment into water, food, and tissue, studies like this one sharpen the scientific case for that transition. The genetic alarm bells rung by OGG1 and HPRT1 in these kidney cells are a reminder that the cost of convenience chemistry is not always visible on the label, and that the molecules holding our plastics together may be prying apart something far more delicate inside our cells.

Subject of Research: Cyto-genotoxic effects of the plasticizer dibutyl phthalate in bovine kidney epithelial cells

Article Title: The cyto-genotoxic potential of dibutyl phthalate promotes DNA damage and cellular stress with altered DNA repair gene expression in Madin-Darby Bovine Kidney (MDBK) cells

Article References: Ali, M. M., Ullah, A., Firyal, S., Akhtar, R., Awan, F., Ataya, F. S., Mehmood, H., Raza, S., Majeed, K. A., & Fouad, D. (2026). The cyto-genotoxic potential of dibutyl phthalate promotes DNA damage and cellular stress with altered DNA repair gene expression in Madin-Darby Bovine Kidney (MDBK) cells. BMC Pharmacology and Toxicology. https://doi.org/10.1186/s40360-026-01239-4

Image Credits: AI Generated

DOI: 10.1186/s40360-026-01239-4

Keywords: dibutyl phthalate, phthalates, genotoxicity, DNA damage, MDBK cells, comet assay, micronucleus, OGG1, HPRT1, endocrine disruptors, plasticizers, genomic instability