Received: 2 May 2019

Revised: 21 July 2019

Accepted: 26 July 2019

DOI: 10.1002/hon.2659

LETTER TO THE EDITOR

A pilot assessment of xanthine oxidase activity in plasma frompatients with hematological malignancies using a highly
sensitive assay
Xanthine oxidoreductase (XOR) is the key enzyme in the production

excluded. Alternatively, plasma XO activity was measured after theof uric acid. XOR is abundantly expressed as xanthine dehydrogenase

discontinuation of XO inhibitors.

(XDH) mainly in the liver and intestine. XDH is physiologically

Statistical analysis was performed using a standard software pack-converted to xanthine oxidase (XO) by proteases on the vasculatureage (JMP version 14; SAS Institute Inc, Cary, North Carolina). All datain the blood. Noticeably, when liver is suffered from hypoxia/inflam-

were examined for normality by Shapiro-Wilk test, and logarithmic

mation, XDH is released into circulation and is subsequently

transformation of variables was performed, where applicable. Contin-

converted into XO.1 Elevated plasma XO level has been reported inuous variables were compared with the Wilcoxon rank sum test. Thecorrelation between variables was calculated using Pearson correla-2,3familial hypercholesterolaemia and cardiovascular diseases.

Regarding hematological malignancies, high value of XO activity inplasma has been reported in patients with non-Hodgkin lymphomation coefficient or Spearman rank correlation coefficient. P values lessthan.05 were considered statistically significant.

and acute lymphocytic leukemia. Allogeneic hematopoietic stem cell

Among enrolled subjects, XO activities in plasma were assessed intransplantation (allo-HSCT) effectively eradicates a line of hematologi-five patients with hematological malignancies before receiving any

4

cal malignancies, but graft-versus-host disease (GVHD) remains lethal
in some cases. However, there seems no validated predictive bio5

marker for acute GVHD. Of note, susceptible organs of GVHD suchas 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 hematologicalmalignancies, we performed an assessment of plasma XO activity inthe 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.

chemotherapies. There were no apparent differences in plasma XO
activities between five patients with hematological malignancies andfive healthy volunteers (Table S2). Figure 1 (A) summarizes the valueof plasma XO activity and clinical manifestations in four patients whounderwent allo-HSCT (cases 1-4). All four cases developed grade 1 or
2 acute GVHD with skin symptoms such as erythema and diarrhea. Incases 1 and 2, there was no increase in plasma XO activity during theclinical course. Notably, in cases 1 and 2, no apparent changes wereobserved in liver transaminases (AST and ALT). On the other hand, incases 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 whoreceived chemotherapy (cases 5-10). No appreciable elevations inThe present study was conducted in accordance with the Helsinki
Declaration.

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

Plasma XO activity was determined using a fluorometric assay
measuring the conversion of pterin to isoxanthopterin with a consid-activity and liver transaminase (AST and ALT) were tightly associatedand concomitantly elevated. Figure 2 (A) shows correlations between

erably high sensitivity.6 (The details of measurement of XO activity inplasma XO activity and a line of clinical parameters in all of blood sam-plasma are shown in Data S1.)

ples (n = 83) from 10 patients analyzed in the present study. For a

Thirty-five patients with hematological malignancies who received

series of clinical parameters correlated with the value of plasma XO

treatment at Ryukyu University Hospital during the period from Octo-

activity shown in Figure 2 (A), analysis using interquartile range (IQR)

ber 2016 to August 2017 were enrolled in this study. Among thewas also carried out to further confirm whether such a correlation

patients studied, in-depth assessments were performed for 10

was similarly observed within intraindividual variations. IQR reflects

patients whose plasma XO activity and clinical parameters were mea-

physiological stability as well as pathological fluctuation in each case.

sured consecutively at least five times during the study period. Profile

Figure 2 (B) clearly shows a significantly positive correlation betweenof patients is summarized in Table S1. To examine the possible differ-plasma XO activity and liver transaminase even in the IQR analyses.

ence in the value of plasma XO activity between patients with hema-The major findings in the present study are as follows. In patients

tological malignancies and healthy subjects, we enrolled five healthy

with hematological malignancies, the value of plasma XO activity wasvolunteers. Patients who routinely use XO inhibitors were carefullytightly associated with that of serum level of liver transaminases (AST

Hematological Oncology. 2019;37:527–530.

wileyonlinelibrary.com/journal/hon© 2019 John Wiley & Sons, Ltd.

527

528

LETTER TO THE EDITORF I G U R E 1 (A), Serial
measurements of clinicalparameters in four cases withallogeneic hematopoietic stem
cell transplantation. After theengraftment, all patients showedskin symptoms and intestinal
symptoms as graft-versus-hostdiseases (GVHD). In patients
without apparent changes inserum levels of livertransaminase (AST and ALT),
there was no significant elevationin plasma XO activity (case 1 andcase 2). On the other hand, incase 3 and case 4, plasma XO
activity and serum levels of livertransaminase (AST and ALT) wereconcomitantly elevated. Blackcircles, diamonds, triangles, andcrosses in the figure indicate
xanthine oxidase activity (XOA),
aspartate aminotransferase (AST),
alanine aminotransferase (ALT),and soluble form of interleukin-2
receptor (sIL-2R), respectively.
The arrow line indicates theperiod of symptoms. The broken
line indicates skin symptoms, andthe solid line indicates diarrhea.
(B), Serial measurements ofclinical parameters in six cases
treated with chemotherapy. In
patients without apparent
elevation in serum levels of livertransaminase (AST and ALT) (case5, case 6, and case 9), there wasno significant elevation in plasma
XO activity. On the other hand,plasma XO activity value andserum levels of livertransaminase (AST and ALT) wereconcomitantly elevated in case 7,
case 8, and case 10. Black circles,
diamonds, triangles, and crosses
in the figure indicate XOA, AST,ALT, and sIL-2R, respectively.
Arrows indicate chemotherapies

and ALT) in the course of allo-HSCT and chemotherapies, suggesting

detectable range.8,9 To overcome this issue, in the present study, wethat the value of plasma XO activity may reflect liver damage because

measured XO activities using considerably high sensitive fluorometricof chemotherapies and related clinical interventions. Plasma XO activ-assay, thereby precisely estimating the plasma XO activities in all sam-ities in humans are considerably low as compared with those inples studied. Our recent study demonstrated that the value of plasma

rodents.7 Therefore, the sensitivity of the measurement is critical toXO activity is not affected by the time point, diet, or exercise.10 The

precisely evaluate the value of XO activity. However, the measure-

IQR analyses in the present study further confirmed that plasma XO

ment methods of previous reports have shown difficulties in theactivity was stable in each individual as far as levels of liver529

LETTER TO THE EDITORF I G U R E 2 (A), Correlation
between the level of plasma XO
activity and a line of clinicalparameters. Data were analyzed
by Spearman rank correlation
coefficient. P values of less than0.05 were considered asstatistically significant. (B),
Correlation between the level ofplasma XO activity and a line ofclinical parameters via theinterquartile range (IQR) analyses.
The correlation between variables
was calculated using Pearson
correlation coefficient for data
showing normal distribution orlog-normal distribution. Spearman
rank correlation coefficient wasemployed for data that did notshow either normal distribution
or log-normal distribution. P
values of less than 0.05 wereconsidered as statisticallysignificant. ALT, alanine
aminotransferase; AST, aspartate
aminotransferase; CRE,creatinine; CRP, C-reactiveprotein; eGFR, estimatedglomerular filtration rate; LDH,lactate dehydrogenase; sIL-2R,soluble interleukin-2 receptor; TBIL, total bilirubin; UA, uric acid;
XOA, xanthine oxidase activity

transaminases were constant. Taken together, it is therefore reason-

15K19520 and 24591338), Council for Science, Technology and Inno-able to speculate that the value of plasma XO activity would bevation (CSTI), Cross-ministerial Strategic Innovation Promotion Pro-

unique in each individual, potentially providing a novel avenue to esti-gram (SIP), “Technologies for Creating Next-generation Agriculture,mate the responsiveness to therapies or conditions of diseases inForestry and Fisheries,” New Energy and Industrial Technology Devel-patients with hematological malignancies. Because the number ofpatients studied was extremely limited, further extensive studies arerequired to strengthen our findings.

opment Organization (NEDO), project for the formation of life science
network (pharmaceutical field), the promotion project of medical clustering of Okinawa prefecture, and grant from Okinawa prefecture forthe promotion of advanced medicine.

ACKNOWLEDGEMEN TS
This work was supported in part by Grants-in-Aid from Japan Society
for the Promotion of Science (JSPS; KAKENHI grant numbers

CONFLIC T OF INT ER E ST
The authors report no conflict of interest.

530

LETTER TO THE EDITORAUTHOR CONTRIBUTIONS

Noboru Hokama and Takashi Shirakura contributed equally to thiswork.

N. Hokama and S. Sunagawa designed the present study, collected
the data, and performed statistical analysis. T. Shirakura designed thestudy and reviewed the manuscript. S. Morishima, S. Nakachi, and Y.
Nishi recruited patients, conducted laboratory tests, and reviewed themanuscript. T. Shirakura, C. Matsui, N. Hase, Y. Murayama, and M.

Peer Review
The peer review history for this article is available at https://publons.
com/publon/10.1002/hon.2659

Tamura performed assays for xanthine oxidase activity. S. Okamoto,
M. Shimabukuro, and K. Nakamura reviewed the manuscript and provided a series of invaluable advices. H. Masuzaki designed the present
study and wrote the manuscript. All authors contributed to the entire
process of manuscript preparation and approved the final version. We
thank I. Nomura, C. Horiguchi, and T. Ikematsu for technical assistance. We are also grateful to M. Hirata, I. Asato, H. Kaneshiro, T.
Uema, and C. Noguchi for secretarial assistance.

ORCID
Noboru Hokama

https://orcid.org/0000-0003-1096-5810
Noboru Hokama1,2
Takashi Shirakura3
Sumito Sunagawa1
Satoko Morishima1
Sawako Nakachi1
Yukiko Nishi1
Yuko Murayama1
Chieko Matsui3
Naoki Hase3
Mizuho Tamura3
Shiki Okamoto1
Michio Shimabukuro4
Katsunori Nakamura2
Hiroaki Masuzaki1

1

Division of Endocrinology, Diabetes and Metabolism, Hematology,Rheumatology (Second Department of Internal Medicine), Graduate
School of Medicine, University of the Ryukyus, Okinawa, Japan
2Department of Hospital Pharmacy, Faculty of Medicine, University ofthe Ryukyus, Okinawa, Japan
3Pharmacology Research Department, Teijin Pharma Limited, Tokyo,Japan
4

Department of Diabetes, Endocrinology and Metabolism School ofMedicine, Fukushima Medical University, Fukushima, JapanRE FE RE NCE S
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SUPPORTING INF ORMATION
Additional supporting information may be found online in theSupporting Information section at the end of this article.