High-dose methotrexate is used in treatment regimens for acute lymphoblastic leukemia, lymphoma and other malignancies, but it has a narrow therapeutic window and can produce severe adverse effects. The drug is excreted unchanged through the kidneys, while a small portion is metabolized in the liver to 7-hydroxymethotrexate, which is also cleared renally but more slowly. In acidic environments, 7-hydroxymethotrexate has only one-third to one-fifth the solubility of methotrexate, making precipitation in renal tubules more likely. Such a process can cause renal injury, which in turn reduces methotrexate clearance and prolongs exposure to toxic concentrations. Oxidative stress and inflammatory responses are also associated with methotrexate-related liver and kidney damage, providing a rationale for examining the redox forms of homocysteine rather than focusing only on their combined concentration.

The researchers enrolled patients with acute lymphoblastic leukemia or lymphoma who received high-dose methotrexate between June 2024 and August 2025. Pediatric patients received doses of 2–5 grams per square metre under Chinese guidance, while adult regimens ranged from 1–5 grams per square metre, with 5 grams per square metre applicable to T-cell acute lymphoblastic leukemia. Methotrexate concentrations were monitored at 42 hours, and leucovorin rescue doses were adjusted accordingly. Rescue was given every six hours until methotrexate fell below specified concentrations, while hydration and urine alkalinization were used to support elimination. All participants had normal hepatic and renal indicators before treatment.

Blood was collected at 42, 72 and 96 hours after methotrexate administration, producing 310 samples from 89 patients: 38 adults and 51 children. Some patients contributed repeated samples within one treatment cycle or across different cycles. There were 143 samples at 42 hours, 110 at 72 hours and 57 at or beyond 96 hours. The number of renal-toxicity events at those times was 12, 17 and 11, respectively. Following the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0, nephrotoxicity was defined as creatinine above the laboratory upper limit of normal. Hepatotoxicity required alanine aminotransferase above that limit together with either aspartate aminotransferase or total bilirubin above the limit. Laboratory abnormalities meeting these definitions and corresponding to at least grade 1 were classified as events.

Total homocysteine was measured in plasma with an enzymatic cycling method. The investigators separately quantified free reduced homocysteine and homocystine using ultrafiltration and HPLC-MS/MS. N-ethylmaleimide was added during sample preparation to prevent thiol auto-oxidation, an important analytical step because free reduced homocysteine is susceptible to conversion into its oxidized form. The assays were validated for calibration range, linearity, lower limits of quantification, precision, accuracy, recovery, matrix effects and stability. The lower limits were 0.05 micromoles per litre for the reduced-form assay and 0.004 micromoles per litre for homocystine; intra-day and inter-day precision had relative standard deviations below 10%, and matrix-effect coefficients of variation were also below 10%. The Hcy/HHcy concentration ratio was calculated as an indirect indicator of the relative redox state of free homocysteine.

Generalized estimating equations were used to account for repeated observations and examine associations between homocysteine measures and indicators of liver or kidney function. Models were adjusted for sex, age, height, body weight, baseline renal and hepatic indicators, methotrexate concentration, methotrexate dose and delayed elimination. False-discovery-rate correction was applied to multiple comparisons, and pediatric and adult patients were analyzed separately. Among children, Hcy/HHcy was significantly negatively associated with creatinine, with a regression coefficient of −0.007, a 95% confidence interval of −0.012 to −0.002 and an adjusted P value of 0.028. Total homocysteine was also negatively associated with alanine aminotransferase and total bilirubin in this age group. Its association with direct bilirubin had an FDR-adjusted P value of 0.065 and therefore did not meet the study’s corrected threshold for statistical significance. No significant associations between the homocysteine measures and hepatic or renal indicators were detected in adults.

Compared with patients whose kidney function remained within the defined laboratory range, those classified as having kidney injury had significantly different concentrations of reduced homocysteine and homocystine at 42 hours, with FDR-adjusted P values of 0.039 and 0.003, respectively. Homocystine was elevated in the kidney-injury group at that time. Reduced homocysteine differed between the groups at 72 hours, with an adjusted P value of 0.003, and homocystine differed at or beyond 96 hours, with an adjusted P value below 0.001. No significant differences in total homocysteine, reduced homocysteine, homocystine or the Hcy/HHcy ratio were found between the liver-injury and normal-function groups after adjustment. The clearest observed group differences therefore involved renal injury, but the observational comparisons do not establish that changes in homocysteine redox forms caused the kidney damage.

Exploratory receiver operating characteristic analyses examined how well the markers distinguished organ injury. Homocystine at 42 hours showed the strongest reported discrimination for renal injury, with an AUC of 0.817, a 95% confidence interval of 0.702–0.932 and a P value below 0.001. At the selected threshold of 0.04 micromoles per litre, sensitivity was 66.7% and specificity was 87.0%. Total homocysteine at 42 hours had an AUC of 0.681, with a 95% confidence interval of 0.492–0.871. The original AUC for Hcy/HHcy was 0.232, indicating that lower ratios were associated with renal injury; reversing the direction of that index produced an adjusted AUC of 0.768, with a 95% confidence interval of 0.628–0.908 and a P value of 0.002. Total homocysteine continued to discriminate renal injury at 72 hours and at or beyond 96 hours, with AUCs of 0.761 and 0.812, respectively.

By contrast, total homocysteine showed reverse discrimination for hepatotoxicity at 72 hours and at or beyond 96 hours, with AUCs of 0.654 and 0.715, meaning that lower concentrations were associated with liver injury in those analyses. Across all sampled observations, median total homocysteine was 8.53 micromoles per litre at 42 hours, 10.13 at 72 hours and 9.61 at or beyond 96 hours. Median homocystine was 0.017 micromoles per litre at the first two time points and 0.020 at or beyond 96 hours, while the median Hcy/HHcy ratio fell from 36.38 at 72 hours to 19.77 at the later point. These were cross-sectional summaries rather than continuous trajectories measured in every patient, because sample numbers decreased and patient overlap was incomplete across time points.

The authors proposed that methotrexate inhibition of dihydrofolate reductase could interfere with the folate-dependent remethylation of homocysteine to methionine, increasing homocysteine early in treatment. Recovery of the methionine cycle and calcium folinate rescue might then contribute to a later decline in total homocysteine, while continued oxidation of reduced homocysteine could increase homocystine and lower the Hcy/HHcy ratio. This mechanism remains hypothetical because oxidative stress was not measured directly. Important limitations further restrict interpretation: baseline homocysteine measurements were unavailable, the cohort was relatively small and clinically heterogeneous, and some potential confounders were not systematically collected. There was no internal validation of the ROC analyses, and the grade 1 toxicity threshold may have included mild or transient laboratory abnormalities. The study therefore provides preliminary evidence about homocysteine redox forms as potential indicators of methotrexate-associated nephrotoxicity, rather than establishing them as validated clinical biomarkers.

Subject of Research: Association of homocysteine redox forms with renal toxicity after high-dose methotrexate in leukemia and lymphoma

Article Title: The relationship of homocysteine and its redox forms with renal toxicity after high-dose methotrexate treatment in patients with leukemia or lymphoma

Article References: Gao, S.-S., Wu, C.-L., Liu, Z.-Y., Zheng, J.-X., Zhou, H.-Y., Xie, J.-Y., Guo, J.-L., & Dong, W.-C. (2026). The relationship of homocysteine and its redox forms with renal toxicity after high-dose methotrexate treatment in patients with leukemia or lymphoma. Frontiers in Pharmacology, 17, Article 1972047. https://doi.org/10.3389/fphar.2026.1972047

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1972047

Keywords: high-dose methotrexate, nephrotoxicity, homocysteine, homocystine, oxidative stress, redox ratio, leukemia, lymphoma, renal function, biomarkers, relationship, redox