A pilot assessment of xanthine oxidase activity in plasma from patients with hematological malignancies using a highly sensitive assay

Xanthine oxidoreductase (XOR) is the key enzyme in the production of uric acid. XOR is abundantly expressed as xanthine dehydrogenase (XDH) mainly in the liver and intestine. XDH is physiologically converted to xanthine oxidase (XO) by proteases on the vasculature in the blood. Noticeably, when liver is suffered from hypoxia/inflammation, XDH is released into circulation and is subsequently converted into XO. Elevated plasma XO level has been reported in familial hypercholesterolaemia and cardiovascular diseases. Regarding hematological malignancies, high value of XO activity in plasma has been reported in patients with non-Hodgkin lymphoma and acute lymphocytic leukemia. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) effectively eradicates a line of hematological malignancies, but graft-versus-host disease (GVHD) remains lethal in some cases. However, there seems no validated predictive biomarker for acute GVHD. Of note, susceptible organs of GVHD such as the liver and intestine are the major production sites of XDH, a biological precursor of XO. In this context, to explore the clinical implication of plasma XO activity in patients with hematological malignancies, we performed an assessment of plasma XO activity in the clinical course of patients with hematological malignancies. The present study was approved by the institutional ethical committee (approved number: 992, Institutional Ethical Committee of University of the Ryukyus on 2 September 2016). Written informed consent was obtained from all participants. The present study was conducted in accordance with the Helsinki Declaration. Plasma XO activity was determined using a fluorometric assay measuring the conversion of pterin to isoxanthopterin with a considerably high sensitivity. (The details of measurement of XO activity in plasma are shown in Data S1.) Thirty-five patients with hematological malignancies who received treatment at Ryukyu University Hospital during the period from October 2016 to August 2017 were enrolled in this study. Among the patients studied, in-depth assessments were performed for 10 patients whose plasma XO activity and clinical parameters were measured consecutively at least five times during the study period. Profile of patients is summarized in Table S1. To examine the possible difference in the value of plasma XO activity between patients with hematological malignancies and healthy subjects, we enrolled five healthy volunteers. Patients who routinely use XO inhibitors were carefully excluded. Alternatively, plasma XO activity was measured after the discontinuation of XO inhibitors. Statistical analysis was performed using a standard software package (JMP version 14; SAS Institute Inc, Cary, North Carolina). All data were examined for normality by Shapiro-Wilk test, and logarithmic transformation of variables was performed, where applicable. Continuous variables were compared with the Wilcoxon rank sum test. The correlation between variables was calculated using Pearson correlation coefficient or Spearman rank correlation coefficient. P values less than.05 were considered statistically significant. Among enrolled subjects, XO activities in plasma were assessed in five patients with hematological malignancies before receiving any chemotherapies. There were no apparent differences in plasma XO activities between five patients with hematological malignancies and five healthy volunteers (Table S2). Figure 1 (A) summarizes the value of plasma XO activity and clinical manifestations in four patients who underwent allo-HSCT (cases 1-4). All four cases developed grade 1 or 2 acute GVHD with skin symptoms such as erythema and diarrhea. In cases 1 and 2, there was no increase in plasma XO activity during the clinical course. Notably, in cases 1 and 2, no apparent changes were observed in liver transaminases (AST and ALT). On the other hand, in cases 3 and 4, the values of both plasma XO activity and liver transaminase (AST and ALT) were tightly associated and concomitantly elevated. Figure 1 (B) summarizes plasma XO activity in six patients who received chemotherapy (cases 5-10). No appreciable elevations in plasma XO activity, AST and ALT, were observed in cases 5, 6, and 9. On the other hand, in case 7, 8, and 10, the values of both plasma XO activity and liver transaminase (AST and ALT) were tightly associated and concomitantly elevated. Figure 2 (A) shows correlations between plasma XO activity and a line of clinical parameters in all of blood samples (n = 83) from 10 patients analyzed in the present study. For a series of clinical parameters correlated with the value of plasma XO activity shown in Figure 2 (A), analysis using interquartile range (IQR) was also carried out to further confirm whether such a correlation was similarly observed within intraindividual variations. IQR reflects physiological stability as well as pathological fluctuation in each case. Figure 2 (B) clearly shows a significantly positive correlation between plasma XO activity and liver transaminase even in the IQR analyses. The major findings in the present study are as follows. In patients with hematological malignancies, the value of plasma XO activity was tightly associated with that of serum level of liver transaminases (AST Received: 2 May 2019 Revised: 21 July 2019 Accepted: 26 July 2019