ORIGINAL ARTICLE

Activity of xanthine oxidase in plasmacorrelates with indices of insulin resistance andliver dysfunction in patients with type 2
diabetes mellitus and metabolic syndrome: A
pilot exploratory study
Sumito Sunagawa1,† , Takashi Shirakura2,†, Noboru Hokama1, Chisayo Kozuka1,3, Masato Yonamine1, Toyotaka Namba1,
Satoko Morishima1, Sawako Nakachi1, Yukiko Nishi1, Tomomi Ikema1, Shiki Okamoto1, Chieko Matsui2, Naoki Hase2,
Mizuho Tamura2, Michio Shimabukuro4, 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, 2Teijin Pharma Limited, Tokyo, Japan, 3Joslin Diabetes Center, Harvard University, Boston, MA, USA, and 4Department of Diabetes, Endocrinology and Metabolism
School of Medicine, Fukushima Medical University, Fukushima, Japan

Keywords
Insulin resistance, Uric acid, Xanthineoxidase*Correspondence
Hiroaki Masuzaki
Tel.: +81-98-895-1147
Fax: +81-98-895-1415
E-mail address:
hiroaki@med.u-ryukyu.ac.jp
J Diabetes Investig 2019; 10: 94–103
doi: 10.1111/jdi.12870

ABSTRACT
Aims/Introduction: There is controversy as to whether hyperuricemia is an independent risk factor for cardiometabolic diseases. The serum level of uric acid is affected by awide variety of factors involved in its production and excretion. In contrast, evidence has
accumulated that locally- and systemically-activated xanthine oxidase (XO), a rate-limiting
enzyme for production of uric acid, is linked to metabolic derangement in humans androdents. We therefore explored the clinical implication of plasma XO activity in patientswith type 2 diabetes mellitus and metabolic syndrome (MetS).
Materials and Methods: We enrolled 60 patients with type 2 diabetes mellitus and
MetS. MetS was defined according to the 2005 International Diabetes Federation guidelines. Plasma XO activity was measured by highly-sensitive fluorometric assay measuring
the conversion of pterin to isoxanthopterin, and explored associations between the valueof plasma XO activity and metabolic parameters.
Results: The value of plasma XO activity was correlated with indices of insulin resistanceand the level of circulating liver transaminases. In contrast, the level of serum uric acidwas not correlated with indices of insulin resistance. The value of plasma XO activity was
not correlated with the serum uric acid level.
Conclusions: Plasma XO activity correlates with indices of insulin resistance and liver
dysfunction in Japanese patients with type 2 diabetes mellitus and MetS. Through assessing the plasma XO activity, patients showing normal levels of serum uric acid with higher
activity of XO can be screened, thereby possibly providing a clue to uncovering metabolicrisks in type 2 diabetes mellitus and MetS patients.

INTRODUCTION
It has long been recognized that hyperuricemia is a potential
risk for a variety of cardiometabolic diseases1,2. In contrast,some previous reports showed that hyperuricemia is not an†

These authors contributed equally to this work.
Received 22 February 2018; revised 4 May 2018; accepted 31 May 201894

J Diabetes Investig Vol. 10 No. 1 January 2019independent risk, but just a surrogate marker for them3,4. Collectively, there is still controversy as to whether hyperuricemia
is an independent risk factor for cardiometabolic diseases. In
contrast, it has been shown that oxidative stress produced byxanthine oxidoreductase (XOR) is related to vascular endothelial dysfunction in humans5 and rodents6. XOR is also a key
enzyme in the production of uric acid (UA), one of the mostª 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution andreproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

http://wileyonlinelibrary.com/journal/jdi

powerful circulating anti-oxidants7. The gene for XOR is initially transcribed as xanthine dehydrogenase (XDH), which
controls purine metabolism mainly in the liver and intestine inhumans8. Previous studies showed that the majority of XOR
protein is derived from the liver throughout the body inhumans8, and noticeably, when the liver is suffers from hypoxia
or inﬂammation, XDH is released into the circulation and is
subsequently converted into xanthine oxidase (XO) by proteases
in the peripheral vasculature9. Importantly, XO locally produces
a considerable amount of reactive oxygen species (ROS), suchas H2O2 and O2-8,10.
It was reported that XO activity in the vasculature is elevatedconsiderably in patients with coronary artery diseases11. Furthermore, multiple regression analyses showed that the value ofplasma XO activity is associated with cardiovascular events andits future risk in patients with chronic heart failure12. It has also
been suggested that local XO activities in a variety of organs
and tissues are likely to elevate under a line of stimuli including
hypoxia, inﬂammatory cytokines and glucocorticoids10,13.
Therefore, there is a possibility that local XO activities in a variety of organs or tissues would be elevated in patients with type2 diabetes and obesity syndrome. However, little is known
about plasma XO activities in the pathophysiology of insulin
resistance and obesity in humans. In this context, here we
explored the clinical implication of plasma XO activity in Japanese patients with type 2 diabetes mellitus and metabolic syndrome (MetS).

METHODS
Participants

The present study was approved by the institutional ethicalcommittee (approval number: 909, institutional ethical committee of University of the Ryukyus on 2 October 2015). Written
informed consent was obtained from all participants. A line ofstudies was carried out in accordance with the Helsinki
Declaration.
We enrolled 50 patients with type 2 diabetes mellitus and
MetS, and ﬁve lean patients without diabetes who did not havehyperuricemia, who were primarily diagnosed as essential
hypertension, primary aldosteronism and non-functional benign
pituitary tumor, respectively. Patients were hospitalized at orvisited the outpatient clinics of the Ryukyu University Hospital,
Okinawa, Japan. In the present study, MetS was deﬁned
according to the 2005 International Diabetes Federation guidelines14. The present study was carried out between May 2016
and December 2017. We consecutively measured plasma XO
activity in the patients recruited, and explored possible associations between the value of plasma XO activity and a variety of
metabolic parameters. Insulin and oral drugs for hyperuricemia
were not used for all patients.

ORIGINAL ARTICLE
Plasma XO activities in T2DM and MetS

collection tubes were purchased from TERUMO Corporation(Venoject II vacuum blood collection tube; Tokyo, Japan). Samples were centrifuged at 1,500 g for 15 min at 4°C for isolationof plasma. Centrifuge was carried out within 1 h after bloodcollection to avoid the leak of hypoxanthine from erythrocytes
into plasma. Supernatants were subsequently transferred to newtubes and were pooled at -80°C as plasma samples.
Measurement of plasma XO activity

Plasma XO activity was determined using a ﬂuorometric assay
measuring the conversion of pterin to isoxanthopterin with aconsiderably high sensitivity15. Brieﬂy, the enzyme reaction was
initiated by mixing 50 lL of plasma with 50 lL of 0.05 mol/L
Tris-HCl buffer (pH 9.0) containing 100 lmol/L pterin and 1%
dimethylsulfoxide. For the blank sample, 0.05 mol/L Tris-HCl
buffer (pH 9.0) was used instead of plasma. After incubation for
3 h at 37°C, 100 lL of 4% HClO4 was added to stop the reaction. The resulting mixture was vigorously shaken and centrifuged at 15,000 g for 10 min at room temperature. Then,
150 lL of the supernatant was neutralized with 6 lL of 5 mol/L
K2CO3 and centrifuged at 15,000 g for 10 min at room temperature; 10 lL of supernatant was subjected ﬂuorometric analysis.
Isoxanthopterin concentration in each sample was measured
by high-performance liquid chromatography with a ﬂuorescence detector. The high-performance liquid chromatographysystem was Nexera X2 (Shimadzu, Kyoto, Japan) and ﬂuorescence detector RF-20Axs (Shimadzu) using a YMC-Triart C18
3-lm column (YMC, Kyoto, Japan). The mobile phase was
20 mmol/L potassium phosphate buffer, the ﬂow-rate was
1.0 mL/min, and the excitation and emission wavelengths were
345 and 410 nm, respectively. For standard solution, 10–
2,000 nmol/L of isoxanthopterin solution diluted with 0.1 N
NaOH or 0.05 mol/L Tris-HCl buffer (pH 9.0) was used. The
area under the curve in the plasma sample and blank sample
was calculated, and the concentration was calibrated by standard linear regression. Isoxanthopterin concentration producedby plasma was given by subtracting the concentration of thesample from that of the blank. Plasma XO activity was
expressed as pmol isoxanthopterin/min/mL (pmol IXP/
min/mL).
Assessment of possible diurnal variation in plasma XO activity

To examine the possible changes in plasma XO activity
throughout the day, we enrolled ﬁve lean patients without diabetes who did not have hyperuricemia, who were primarily
diagnosed as essential hypertension, primary aldosteronism andnon-functional benign pituitary tumor, respectively. Plasma XO
activity was measured at the time-points of 08.00, 15.00 and
23.00 hours, and 08.00 hours the next day.

Sample preparation for assessing XO activity in plasma

Assessment of plasma XO activity before and after the mealintake or 1 h of mild aerobic exercise

Blood was collected into 5-mL tubes containing heparin fromparticipants in a fasting state early in the morning. Blood

To examine the variability of plasma XO activity by having
meals or doing exercise, we newly enrolled 10 patients whoª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

J Diabetes Investig Vol. 10 No. 1 January 201995

ORIGINAL ARTICLE
Sunagawa et al.

were not receiving insulin or oral drugs for hyperuricemia.
Plasma XO activity was measured before/after breakfast andbefore/after lunch (n = 5), as well as before/after ~1 h of mild
aerobic exercise in another group (n = 5). All patients were
provided standardized hospital diets three times a day (25–
30 kcal/kg ideal bodyweight/day). Five patients underwent mild
exercise therapies for 1 h after lunch, including slow pace walking and stretching under the supervision of specialized medical
doctors. The heart rate of participants was strictly controlled to<120 b.p.m. during the course of exercise.
Measurement of metabolic parameters

Anthropometric variables were measured in the standing position. Body mass index (BMI) was calculated as weight (in kg)
divided by the square of height in meters. Serum biochemicalvariables including aspartate aminotransferase (AST), alanine
aminotransferase (ALT), c-glutamyltransferase, triglyceride,
high-density lipoprotein cholesterol, low-density lipoproteincholesterol, creatinine, fasting blood glucose and immunoreactive insulin (IRI) were measured with a conventional automatedanalyzer. High-sensitivity C-reactive protein (hs-CRP) was measured by using nephelometry, a latex particle enhanced
immunoassay (Behring Nephelometer II; Dade Behring Marburg GmbH, Marburg, Germany). High-molecular weight adiponectin was measured by electro-chemiluminescent enzyme
immunoassay (Lumipulse Presto II, Fujirebio Inc., Tokyo,
Japan) at SRL Inc. (Tokyo, Japan).
Multiple regression analyses to explore possible independent
factors for directly inﬂuencing the plasma XO activity

A multiple linear regression analysis with plasma XO activity as
a dependent variable, and sex (female = 1, male = 0), age, BMI,
glycated hemoglobin (HbA1c), UA, estimated glomerular ﬁltration rate (eGFR), natural logarithm (ln) AST, ln ALT, fasting
IRI and homeostatic model assessment for insulin resistance(HOMA-IR) as explanatory variables was carried out by using
the least squared estimation. Because the serum value of AST
and ALT, as well as the plasma value of XO activity, did notshow a normal distribution, each value was converted logarithmically (ln AST, ln ALT and ln plasma XO activity, respectively), and subjected to a series of multiple regression analyses.
In all the models developed (Table 2; model 1, 2 and 3), ln
plasma XO activity was included as a dependent variable, andsex, age, BMI, HbA1c, UA and eGFR as common explanatory
variables. We then carried out analyses by using ln AST, ln
ALT and fasting IRI as independent variables (Table 2, model1) and ln AST, ln ALT and HOMA-IR as independent variables (Table 2, model 2), as well as HOMA-IR as an independent variable (Table 2, model 3).
Statistical analysis

Data are expressed as mean – standard error of the mean ormean – standard deviation, where applicable. For a line ofanalyses, plasma XO activity, AST and ALT were natural
96

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logarithm transformed (ln) to normalize the skewed distribution. Statistical calculations for signiﬁcant differences were carried out using the Wilcoxon signed-rank test, Wilcoxon rank
sum test and Spearman’s rank correlation coefﬁcients. Signiﬁcance was accepted at P < 0.05. Statistical analyses were carriedout using standard software packages (JMP version 12; SAS
Institute Inc., Cary, NC, USA).

RESULTSClinical characteristics of participants

The clinical characteristics of participants (n = 50) in analysesof plasma XO activity in relation to metabolic parameters RE
summarized in Table 1. Their mean age was 55 – 14 years andmean BMI was 29.5 – 6.2 kg/m2. The numbers of patients
who had used antidiabetic drugs were 12 (sulfonylureas or glinides), 26 (dipeptidyl peptidase-4 inhibitors), 25 (metformin),four (a-glucosidase inhibitors), four (thiazolidines) and three(sodium–glucose cotransporter 2 inhibitors).
Characterization of XO activity in plasma

The value of plasma XO activity was distributed across a wide
range (0.05–5.40 pmol IXP/min/mL), with roughly 100-foldvariations from minimum to maximum levels in patients with
type 2 diabetes mellitus and MetS (Figure 1). It is recognizedthat plasma XO activities in humans show considerably low
values compared with those in rodents16–18, and therefore, ahighly sensitive method to precisely detect the XO activities isrequired. In the present study, we measured XO activities using
highly sensitive ﬂuorometric assay, so we could detect the XO
activities in all samples. We conﬁrmed that the XO activities
measured were completely blocked by febuxostat, which is apotent, selective XO inhibitor, thereby showing that we could
determine the value of XO activities with high sensitivity andspeciﬁcity.
In contrast, the values of serum UA were distributed in a relatively narrow range (2.4–9.2 mg/dL), with roughly fourfold
variations from minimum to maximum levels (Figure 1;
Table 1). Consistent with a previous report1, the mean value ofserum UA in men (6.0 mg/dL) showed a trend of being higher
than that in women (5.6 mg/dL; Figure 1, left side; Table 1).
Notably, there was no statistical difference in plasma XO activity between men and women (Figure 1, right side; Table 1).
Value of plasma XO activity throughout the day, before/after
meals or mild aerobic exercise in lean patients without
diabetes

To examine whether plasma XO activity shows diurnal variation, we measured plasma XO activity at 08.00, 15.00 and
23.00 hours, and 08.00 hours the next day in ﬁve lean patients
without diabetes. There were no statistical changes in the valueof plasma XO activity among the four time-points (Figure 2a).
To examine whether plasma XO activity was affected bymeals or exercise, we measured plasma XO activity before/after
breakfast or lunch, as well as before/after 1 h of mild aerobicª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

ORIGINAL ARTICLE
Plasma XO activities in T2DM and MetS

http://wileyonlinelibrary.com/journal/jdi

Table 1 | Clinical characteristics of the study participantsn (women:men)Age (years)
BMI (kg/m2)
HbA1c (%)
FPG (mg/dL)
FIRI (lIU/mL)
HOMA-IR
c-GTP (IU/L)Cre (mg/dL)
eGFR (mL/min/1.73 m2)
TG (mg/dL)
HDL-C (mg/dL)
LDL-C (mg/dL)
UA, women + men (mg/dL)
UA, women (mg/dL)
UA, men (mg/dL)
ln AST (IU/L)
ln ALT (IU/L)
ln XO activity, women + men(pmol IXP/min/mL)
ln XO activity, women(pmol IXP/min/mL)
ln XO activity, men(pmol IXP/min/mL)

50 (24:26)
55 – 14 (22–79)
29.5 – 6.2 (16.1–53.5)
7.8 – 1.7 (5.4–12.3)
132 – 34 (77–241)
11.1 – 6.9 (1.8–34.2)
3.4 – 2.1 (0.66–9.71)
42 – 29 (10–161)
0.7 – 0.2 (0.4–1.2)
85 – 23 (40–138)
172 – 81 (59–417)
45 – 11 (23–73)
108 – 33 (41–168)
5.8 – 1.5 (2.4–9.2)
5.6 – 1.7 (2.8–9.2)
6.0 – 1.3 (2.4–7.9)
3.1 – 0.50
3.3 – 0.62
-0.54 – 1.1-0.40 – 1.2-0.67 – 1.0

Data are expressed as mean – standard deviation (range) for normally
distributed values and median (range) for non-normally distributed values. ALT, aspartate aminotransferase; AST, alanine aminotransferase; BMI,
body mass index; Cre, creatinine; eGFR, estimated glomerular filtration
rate; FIRI, fasting immunoreactive insulin; FPG, fasting plasma glucose;c-GTP, c-glutamyltransferase; HbA1c, glycated hemoglobin; HDL-C,
high-density lipoprotein cholesterol; HOMA-IR, homeostatic model
assessment for insulin resistance; IRI, immunoreactive insulin; IXP,
isoxanthopterin; LDL-C, low-density lipoprotein cholesterol; ln, natural
logarithm; TG, triglyceride; UA, uric acid; XO, xanthine oxidase.

exercise. There were no statistical changes in plasma XO activities between before and after the meals (breakfast, lunch) or
mild aerobic exercise (Figure 2b).
Value of plasma XO activity in patients with type 2 diabetes
mellitus and MetS

We assessed plasma XO activity in patients with type 2 diabetes
mellitus and MetS, in comparison with lean patients without
diabetes. Plasma XO activity in patients with type 2 diabetes
mellitus and MetS (n = 50, median value 0.55 pmol IXP/min/mL) was signiﬁcantly higher than that in lean patients without
diabetes (n = 5, median value 0.13 pmol IXP/min/mL,
P = 0.003). We also determined the plasma XO activity inpatients with type 2 diabetes mellitus and in patients without
type 2 diabetes mellitus but with MetS. Plasma XO activity
showed no statistical difference between patients with type 2
diabetes mellitus (n = 43, median value 0.46 pmol IXP/min/mL) and patients without type 2 diabetes mellitus but with MetS
(n = 7, median value 0.83 pmol IXP/min/mL, P = 0.146).

ª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

Correlations between the value of logarithmic plasma XO
activity and metabolic parameters

Correlations between the value of ln plasma XO activity andthat in a line of metabolic markers are shown in Figures 3–6.
Importantly, there was no correlation between ln XO activity
and serum UA (Figure 3a). Serum UA level also showed nocorrelations with parameters of insulin resistance (Figure 3b,c).
Noticeably, the level of ln XO activity was strongly correlatedwith fasting IRI and HOMA-IR (Figure 4e,f). In addition, thelevel of ln XO activity was weakly but signiﬁcantly correlatedwith BMI (Figure 4b). In contrast, ln XO activity was not correlated with age, HbA1c and FPG (Figure 4a,c,d).
To examine whether indices of insulin resistance would be
affected by receiving antidiabetic drugs in the patients studied,
we assessed the correlation between ln XO activity and indicesof insulin resistance in participants not taking metformins or
thiazolidines, or sodium–glucose cotransporter 2 inhibitors(n = 19). In these participants, plasma XO activities were also
correlated with indices of insulin resistance (fasting IRI
r = 0.74, P < 0.001; HOMA-IR r = 0.67, P = 0.001, respectively). In contrast, there was no statistical difference in thevalue of HOMA-IR between patients treated with sulfonylureas
or glinides (n = 12) and other patients (n = 38; P = 0.220).
Notably, the level of ln XO activity was strongly correlatedwith that of ln AST and ln ALT (Figure 5a,b), and was also
weakly but signiﬁcantly correlated with the Fibrosis-4 score19,
one of the representative markers for hepatic ﬁbrosis (r = 0.32,
P < 0.001). The level of plasma XO activity was also signiﬁcantly correlated with that of ln c-glutamyltransferase (Figure 5c), whereas it was not correlated with creatinine and
eGFR (Figure 5d,e), both of which are known to be associatedwith serum UA in patients with chronic kidney diseases.
To explore if ln XO activity would be associated with parameters of inﬂammation, we assessed serum levels of hs-CRP andadiponectin, both of which are representative biomarkers for
systemic inﬂammation (Figure 6). As serum levels of hs-CRP
and adiponectin did not show a normal distribution, they wereconverted logarithmically (ln hs-CRP and ln adiponectin,
respectively). Consequently, there were no signiﬁcant relationships between ln XO activity and ln adiponectin (Figure 6a), as
well as between ln XO activity and ln hs-CRP (Figure 6b).
Multiple regression analyses to explore possible independent
factors for directly inﬂuencing the plasma XO activity

To explore possible independent factors for directly inﬂuencing
the plasma XO activity in patients with type 2 diabetes mellitus
and MetS, we carried out multiple regression analyses (Table 2).
As serum values of AST, ALT and plasma XO activity did notshow a normal distribution, they were converted logarithmically(ln AST, ln ALT and ln plasma XO activity). In a series of analyses, ln XO activity was used as a dependent variable.
When values of age, BMI, sex, HbA1c, UA, eGFR, ln AST,
ln ALT and fasting IRI were used as explanatory variables, thevalue of ln ALT was an independent strong factor for

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Sunagawa et al.

7

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(a)

0.25
XO activity(pmol IXP/min/mL)

6

5
Ratio of UA/mean of UA and
XO activity/mean of XO activity

0.30

4

0.20
0.15
0.10
0.05

3

0.00
8.00

2

15.00

8.00
In the nextday

Time (hours)

1(b) 1.20

0

0.60
0.50
0.40
0.30
0.20

UA
mean /
Fema of UA
le + M
ale
UA/
mean
of U
Fema A
le
UA/
mean
of U
Male A
XO
mean activity/ofFema XO activity
le + Male
XO activity/
mean
of XO
a
Fema ctivityleXOmean activity/of XO
acMale tivity

Figure 1 | Distribution of the values of plasma xanthine oxidase (XO)
activity and serum uric acid (UA) in patients with type 2 diabetes
mellitus and metabolic syndrome. “UA/mean of UA” indicates the ratioof serum UA value vs the mean of serum UA value. “XO activity/meanof XO activity” indicates the ratio of plasma XO activity value vs themean of plasma XO activity value. Small bars in each column show therespective range of standard deviations.

determining plasma XO activity (Table 2, model 1). When values of age, BMI, sex, HbA1c, UA, eGFR, ln AST, ln ALT and
HOMA-IR were used as explanatory variables, the value of ln
ALT was also an independent strong factor for determining theplasma XO activity (Table 2, model 2). Furthermore, when values of age, BMI, sex, HbA1c, UA, eGFR and HOMA-IR wereused as explanatory variables without a line of parameters forliver functions including AST, ALT and c-glutamyltransferase,the value of HOMA-IR was an independent factor for determining the plasma XO activity (Table 2, model 3). These datasuggest that the values of ALT and HOMA index, a clinicalmarker for hepatic insulin resistance, might be independent factors for directly inﬂuencing the plasma XO activity in patientswith type 2 diabetes mellitus and MetS.

DISCUSSION
The major ﬁnding of the present study was that the valueof plasma XO activity was signiﬁcantly correlated with indicesof insulin resistance and liver dysfunction in a small number of
J Diabetes Investig Vol. 10 No. 1 January 2019

XO activity(pmol IXP/min/mL)

1.00

–1

98

23.00

0.80

BE

AE

0.60
0.40
0.20

BB

AB

BL

AL

Figure 2 | The value of plasma xanthine oxidase (XO) activity did notshow apparent diurnal variation and was not altered by meals or
aerobic mild exercise in lean patients without diabetes. (a) In leanpatients without diabetes, plasma XO activity was measured at the
point of 08.00, 15.00 and 23.00 hours, and 08.00 hours in the next day(n = 5). (b) In patients now being treated with insulin or oral drugs for
hyperuricemia, plasma XO activities were measured before/after
breakfast and before/after lunch (large graph, n = 5), as well as before/
after ~1 h of mild aerobic exercise in another group (small graph,n = 5). Data are expressed as mean – standard error of the mean, andwere analyzed by Wilcoxon signed-rank tests. BB, before breakfast; AB,after breakfast; BL, before lunch; AL, after lunch; BE, before exercise; AE,
after exercise; IXP, isoxanthopterin.

Japanese patients with type 2 diabetes mellitus and MetS. In
contrast, the level of serum UA was not correlated with indicesof insulin resistance. Importantly, the value of plasma XO activity was not correlated with the serum UA level. The multiple
regression analyses further suggested that the value of plasma
XO activity would be inﬂuenced by liver dysfunction (ALT)and hepatic insulin resistance (HOMA-IR).
It is widely accepted that hyperinsulinemia followed by systemic insulin resistance causes elevation of serum UA levels
through enhancement of renal proximal tubular sodium reabsorption20. Furthermore, insulin resistance in the liver,ª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

ORIGINAL ARTICLE
Plasma XO activities in T2DM and MetS

http://wileyonlinelibrary.com/journal/jdi

UA (mg/dL)(a) 1098
7

r = 0.23
P = 0.11

6
5
4
3
2
–3

–2

0
–1
In plasma XOA(b) 102

r = 0.12
P = 0.39

98

UA (mg/dL)

8

UA (mg/dL)(c) 10r = 0.15
P = 0.29

9

1

7
6
5

7
6
5

4

4

3

3

2

2
0

5

10

15

20

25

35

30

2

0

4

8

6

10

HOMA-IR

Fasting IRI (µIU/mL)

Figure 3 | Serum uric acid (UA) level did not correlate with (a) plasma xanthine oxidase activity (XOA), (b) immunoreactive insulin (IRI) and (c)homeostatic model assessment for insulin resistance (HOMA-IR) in patients with type 2 diabetes mellitus and metabolic syndrome. Data wereanalyzed by Spearman’s rank correlation coefficients. ln, natural logarithm.

2

2

0
–1
–2
–3

(c)

r = 0.39
P = 0.01

1
In plasma XOA

1
In plasma XOA(b)r = 0.16
P = 0.27

0
–1
–2

30

40

50

60

70

80

15

20

25

(e)

r = 0.11
P = 0.46

30

35

40

45

50

–1
–2

55

5

6

7

2

0
–1
–2
–3

(f)

r = 0.52
P < 0.001

1

200

250

2

9

10

11

12

13

r = 0.51
P < 0.001

1

0
–1
–2
–3

FPG (mg/dL)

8

HbA1c (%)

In plasma XOA

1

In plasma XOA

In plasma XOA

(d) 2

150

0

BMI (kg/m2)

Age

100

r = 0.07
P = 0.65

–3

–3
20

2
1

In plasma XOA

(a)

0
–1
–2
–3

0

5

10

15

20

25

Fasting IRI (µIU/mL)

30

35

0

2

4

6

8

10

HOMA-IR

Figure 4 | Plasma xanthine oxidase activity (XOA) showed positive correlations with indices of insulin resistance in patients with type 2 diabetes
mellitus and metabolic syndrome. Data were analyzed by Spearman’s rank correlation coefficients. BMI, body mass index; FPG, fasting plasma
glucose; HbA1c, glycated hemoglobin; HOMA-IR, homeostatic model assessment for insulin resistance; IRI, immunoreactive insulin; ln, natural
logarithm.

ª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

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Sunagawa et al.

1(b)r = 0.79
P < 0.001

0
–1
–2

1

0
–1
–2
–3

2.5

3

3.5
In AST4

2

r = 0.15
P = 0.31

1

(e)

0
–1
–2

r = 0.46
P < 0.001

0
–1
–2
–3

2

4.5

2.5

3

3.5
In ALT

4

4.5

5

2.5

3

3.5
4
In G-GTP

4.5

52
r = 0.03
P = 0.84

1
In plasma XOA

In plasma XOA(c) 2

r = 0.85
P < 0.001

1

–3

(d)

2

In plasma XOA

2

In plasma XOA

In plasma XOA(a)

http://wileyonlinelibrary.com/journal/jdi

0
–1
–2
–3

–3
0.4

0.6

0.8

1

1.2

CRE (mg/dL)

40

60

80

100 120 140 160 180

eGFR (ml/min/1.73 m2)

Figure 5 | Correlations between natural logarithm (ln) plasma xanthine oxidase activity (XOA) and indices of liver dysfunction or parameters closely
related to circulating uric acid level in patients with type 2 diabetes mellitus and metabolic syndrome. Data were analyzed by Spearman’s rankcorrelation coefficients. AST, alanine aminotransferase; ALT, aspartate aminotransferase; CRE, creatinine; eGFR, estimated glomerular filtration rate; GGTP, γ-glutamyltransferase; ln, natural logarithm.

commonly seen in type 2 diabetes mellitus and MetS, alternatively activates the pentose phosphate pathway, resulting in activation of a de novo synthesis pathway of UA21. Importantly,
the circulating level of UA is inﬂuenced by a variety of factors,
including dehydration, purine- or fructose-rich foods, alcoholand urinary sugar-associated excretion of urate22. In this context, it might be critical to identify patients showing normal
levels of serum UA with higher activity of XO, thereby uncovering hidden risks for type 2 diabetes mellitus and MetS.
In the present study, we showed that the value of plasma
XO activity was correlated with that in liver transaminases(AST, ALT) in patients with type 2 diabetes mellitus and MetS.
Such a positive correlation between plasma XO activity andliver dysfunction is consistent with the notion that leakage of
XDH into the circulation and subsequent conversion of XDH
to XO by proteases in the vasculature is exaggerated by hypoxia23, ischemia/reperfusion24, inﬂammation with tissue damage9
and viral infection25 in the liver. The present ﬁndings are alsoin accordance with the notion that XO is activated by hypoxia,
inﬂammation and animal fat13.
A recent study in young and healthy individuals demonstrated that plasma XO activity was shown to correlate with
BMI, indices of insulin resistance (quantitative insulin sensitivity
check index) and serum levels of UA26. In contrast, the present
study showed that plasma XO activity correlates with insulin
resistance and dysfunction of the liver and BMI, but not with

100

J Diabetes Investig Vol. 10 No. 1 January 2019

serum UA in middle-aged (mean age 55 – 14) patients withestablished type 2 diabetes mellitus and MetS. Although themechanism whereby such a difference could occur between two
studies is not fully understood, the average of age and severityof metabolic diseases would interfere with the results. To clarify
this issue, further extensive studies are warranted. Also, in aprevious report, plasma XO activity in patients with familial
combined hyperlipidemia was correlated with indices of insulin
resistance (HOMA-IR), UA, high-density lipoprotein cholesterol
and inﬂammatory markers (nuclear factor-jB, interleukin-6,
hs-CRP)27. To our knowledge, the present study might be theﬁrst to show that the value of plasma XO activity is correlatedwith indices of insulin resistance and liver dysfunction independently of UA in patients with type 2 diabetes mellitus and
MetS.
Importantly, the sensitivity and speciﬁcity of the methods todetermine the level of plasma XO activity would also interferewith the results, because plasma XO activities in humans show
a considerably low value compared with those in rodents16–18.
It should also be noted that almost all of the previous reports
on plasma XO activity measured UA or its stable isotope produced by XO, and estimated the value as an enzymatic activityof XO. However, such a method resulted in relatively low sensitivities28. Even in recent reports, considerable numbers ofplasma samples were below the detectable range in assays of
XO activity29,30. In other assays, the production of ROS wasª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

ORIGINAL ARTICLE
Plasma XO activities in T2DM and MetS

http://wileyonlinelibrary.com/journal/jdi

(a)

2
1

In XO activity

Table 2 | Multiple regression analysis with natural logarithm xanthineoxidase activity as a dependent variable and metabolic parameters as
explanatory variablesr = 0.12
P = 0.420Model 1Model 2Model 3

–1

Adjusted
r2 = 0.747

Adjusted
r2 = 0.737

Adjusted
r2 = 0.254

–2
–3
–0.5

0

0.5

1

1.5

2

Sex
Age
BMI
HbA1c
UA
eGFR
ln ASTln ALT
FIRI
HOMA-IRIn adiponectin(b)2

r = 0.14
P = 0.33

In XO activity

1
0
–1
–2
–3
4

5

6

7

8

9

10

11

12

In hs-CRP

Figure 6 | Correlations between natural logarithm (ln) plasma xanthineoxidase (XO) activity and (a) ln adiponectin or (b) ln high-sensitivity Creactive protein (hs-CRP) in patients with type 2 diabetes mellitus andmetabolic syndrome. Data were analyzed by Spearman’s rankcorrelation coefficients.

measured for assessing plasma XO activity31. However, ROS
was produced by oxidases other than XO, and was eliminatedby anti-oxidant enzymes, such as superoxide dismutase. It istherefore difﬁcult to precisely measure the XO activity when
ROS is used. In the present study, we measured XO activities
using considerably high sensitivity ﬂuorometric assay, thereby
precisely estimating the plasma XO activities in all samples. We
also conﬁrmed that the XO activities measured were completely
blocked by febuxostat, which is a potent, selective XO inhibitor.
Taken together, in the present study, we were successful indetermining the value of XO activities distributed in a very
wide range.
We acknowledge that there were some limitations in the present study. First, due to the small sample size studied, the present study is deﬁned as a pilot, exploratory study. Second, sucha clinical, observational work as the current study could notclarify the direct mechanistic link between the elevation ofplasma XO activity and pathophysiology of insulin resistance
and/or liver dysfunction. However, previous works in rats
showed that oxidative stress provoked by administration ofxanthine and XO caused the elevation of plasma XO activity
and insulin resistance32, suggesting a causal relationshipbetween the two events. Regarding liver dysfunction, the elevation of plasma XO activity is a reﬂection of liver damage, suchª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

P < 0.001bP-0.051
0.045
0.180
0.108
0.071
0.036
0.188
0.614
0.106

0.582
0.662
0.052
0.212
0.416
0.694
0.314
0.001
0.284

P < 0.001bP

P = 0.006bP-0.046
0.043
0.185
0.080
0.066
0.037
0.171
0.628

0.613
0.677
0.045
0.375
0.451
0.690
0.371
0.001

-0.163-0.0090.297-0.0140.050-0.021

0.281
0.575
0.056
0.921
0.736
0.893

0.105

0.276

0.469

0.002

Multiple linear regression analysis with natural logarithm (ln) xanthineoxidase activity as a dependent variable, and sex (female = 1, male =0), age, body mass index (BMI), glycated hemoglobin (HbA1c), uric acid(UA), estimated glomerular filtration rate (eGFR), ln alanine aminotransferase (AST), ln aspartate aminotransferase (ALT), fasting immunoreactive
insulin (FIRI) and homeostatic model assessment for insulin resistance(HOMA-IR) as explanatory variables was carried out by using the leastsquares estimation. In all the models developed (model 1, 2 and 3),
plasma xanthine oxidase activity was included as a dependent variable,
and sex, age, BMI, HbA1c, UA and eGFR as common explanatory variables. We then carried out analyses by using ln AST, ln ALT, and FIRI as
independent variables (model 1), and ln AST, ln ALT and HOMA-IR as
independent variables (model 2), as well as HOMA-IR as an independent variable (model 3). b, standard partial regression coefficient.

as inﬂammation or hypoxia, which is sometimes related withthe pathophysiology of type 2 diabetes and obesity syndrome.
In this context, further studies are warranted to clarify the possible pathophysiological relationship between the elevation ofplasma XO activity and insulin resistance and/or liver dysfunction in humans. Third, participants diagnosed as type 2 diabetes mellitus and MetS had taken some medications including
oral antidiabetic drugs. This would ﬂuctuate the value of metabolic markers to a certain extent. For example, a previous
report showed that plasma XO activity and thiobarbituric acidreactive substances were decreased after patients with type 2
diabetes mellitus took metformin33. In the present study, however, there was no statistical difference of plasma XO activities
between participants taking metformin and not taking metformin (data not shown). Furthermore, in patients not treatedwith metformin, thiazolidinedione or sodium–glucose cotransporter 2 inhibitors, plasma XO activities were also signiﬁcantly
correlated with indices of insulin resistance (fasting IRI
r = 0.80, P < 0.001; HOMA-IR r = 0.67, P < 0.001, respectively). These results raise a possibility that the current

J Diabetes Investig Vol. 10 No. 1 January 2019101

ORIGINAL ARTICLE
Sunagawa et al.

medication of oral antidiabetic drugs would not strongly inﬂuence plasma XO activities in patients with type 2 diabetes mellitus and Mets, but further prospective studies are warranted totest this hypothesis.
In summary, in the present pilot, exploratory study, the valueof plasma XO activity was signiﬁcantly correlated with indicesof insulin resistance and liver dysfunction in Japanese patientswith type 2 diabetes mellitus and MetS. Plasma XO activity
shows no apparent diurnal variations and no signiﬁcantchanges after meals or mild aerobic exercise. Through assessing
plasma XO activities precisely in clinics, patients showing normal levels of serum UA with higher activity of XO can bescreened, thereby possibly providing a clue to uncover hidden
risks for type 2 diabetes mellitus and MetS.

ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid from theJapan Society for the Promotion of Science (JSPS; KAKENHI
grant numbers 15K19520 and 24591338), Council for Science,
Technology and Innovation (CSTI), Cross-ministerial Strategic
Innovation Promotion Program (SIP), “Technologies for Creating Next-Generation Agriculture, Forestry and Fisheries”, Lotte
Foundation, Japan Foundation for Applied Enzymology, New
Energy and Industrial Technology Development Organization(NEDO), Project for Formation of Life Science Network (Pharmaceutical ﬁeld), the Promotion Project of Medical Clusteringof Okinawa prefecture and a grant from Okinawa Prefecture
for Promotion of Advanced Medicine. We thank Y Murayama
and T Ikematsu for excellent technical help. We are also grateful to M Hirata, I Asato, H Kaneshiro, T Uema and C Noguchi
for excellent secretarial assistance.
DISCLOSURE
The authors declare no conﬂict of interest.
REFERENCES
1. Levine W, Dyer AR, Shekelle RB, et al. Serum uric acid and11.5-year mortality of middle-aged women: findings of the
Chicago Heart Association Detection Project in Industry. J
Clin Epidemiol 1989; 42: 257–267.
2. Fang J, Alderman MH. Serum uric acid and cardiovascularmortality the NHANES I epidemiologic follow-up study,
1971-1992. National Health and Nutrition Examination
Survey. JAMA 2000; 283: 2404–2410.
3. Culleton BF, Larson MG, Kannel WB, et al. Serum uric acidand risk for cardiovascular disease and death: theFramingham Heart Study. Ann Intern Med 1999; 131: 7–13.
4. Moriarity JT, Folsom AR, Iribarren C, et al. Serum uric acidand risk of coronary heart disease: Atherosclerosis Risk
in Communities (ARIC) Study. Ann Epidemiol 2000; 10:
136–143.
5. Battelli MG, Polito L, Bolognesi A. Xanthine oxidoreductase
in atherosclerosis pathogenesis: not only oxidative stress.
Atherosclerosis 2014; 237: 562–567.
102

J Diabetes Investig Vol. 10 No. 1 January 2019http://wileyonlinelibrary.com/journal/jdi

6. White CR, Darley-Usmar V, Berrington WR, et al. Circulatingplasma xanthine oxidase contributes to vascular dysfunction
in hypercholesterolemic rabbits. Proc Natl Acad Sci U S A
1996; 93: 8745–8749.
7. Martin HM, Hancock JT, Salisbury V, et al. Role of xanthineoxidoreductase as an antimicrobial agent. Infect Immun
2004; 72: 4933–4939.
8. Kelley EE. Dispelling dogma and misconceptions regarding
the most pharmacologically targetable source of reactive
species in inflammatory disease, xanthine oxidoreductase.
Arch Toxicol 2015; 89: 1193–1207.
9. Houston M, Estevez A, Chumley P, et al. Binding of
xanthine oxidase to vascular endothelium. Kinetic
characterization and oxidative impairment of nitric oxidedependent signaling. J Biol Chem 1999; 274: 4985–4994.
10. Berry CE, Hare JM. Xanthine oxidoreductase andcardiovascular disease: molecular mechanisms andpathophysiological implications. J Physiol 2004; 555: 589–
606.
11. Spiekermann S, Landmesser U, Dikalov S, et al. Electron spinresonance characterization of vascular xanthine and NAD(P) H oxidase activity in patients with coronary arterydisease: relation to endothelium-dependent vasodilation.
Circulation 2003; 107: 1383–1389.
12. Otaki Y, Watanabe T, Kinoshita D, et al. Association ofplasma xanthine oxidoreductase activity with severity andclinical outcome in patients with chronic heart failure. Int J
Cardiol 2017; 228: 151–157.
13. Tsushima Y, Nishizawa H, Tochino Y, et al. Uric acidsecretion from adipose tissue and its increase in obesity. J
Biol Chem 2013; 288: 27138–27149.
14. Alberti KG, Zimmet P, Shaw J, et al. The metabolic
syndrome-a new worldwide definition. Lancet 2005; 366:
1059–1062.
15. Beckman JS, Parks DA, Pearson JD, et al. A sensitive
fluorometric assay for measuring xanthine dehydrogenase
and oxidase in tissues. Free Radic Biol Med 1989; 6: 607–615.
16. Wajner M, Harkness RA. Distribution of xanthine
dehydrogenase and oxidase activities in human and rabbit
tissues. Biochim Biophys Acta 1989; 991: 79–84.
17. Muxfeldt M, Schaper W. The activity of xanthine oxidase inheart of pigs, guinea pigs, rabbits, rats, and humans. BasicRes Cardiol 1987; 82: 486–492.
18. Abadeh S, Killacky J, Benboubetra M, et al. Purification andpartial characterization of xanthine oxidase from humanmilk. Biochim Biophys Acta 1992; 1117: 25–32.
19. Shah AG, Lydecker A, Murray K, et al. Comparison ofnoninvasive markers of fibrosis in patients withnonalcoholic fatty liver disease. Clin Gastroenterol Hepatol
2009; 7: 1104–1112.
20. Facchini F, Chen YD, Hollenbeck CB, et al. Relationshipbetween resistance to insulin-mediated glucose uptake,urinary uric acid clearance, and plasma uric acidconcentration. JAMA 1991; 266: 3008–3011.

ª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltdhttp://wileyonlinelibrary.com/journal/jdi

21. Leyva F, Wingrove CS, Godsland IF, et al. The glycolytic
pathway to coronary heart disease: a hypothesis.
Metabolism 1998; 47: 657–662.
22. Yamamoto T, Moriwaki Y, Takahashi S. Effect of ethanol on
metabolism of purine bases (hypoxanthine, xanthine, anduric acid). Clin Chim Acta 2005; 356: 35–57.
23. Aslan M, Ryan TM, Adler B, et al. Oxygen radical inhibitionof nitric oxide-dependent vascular function in sickle celldisease. Proc Natl Acad Sci U S A 2001; 98: 15215–15220.
24. Tan S, Gelman S, Wheat JK, et al. Circulating xanthineoxidase in human ischemia reperfusion. South Med J 1995;
88: 479–482.
ski J. Diagnostic value of determination
25. Wolko K, Krawczyn
of serum xanthine oxidase activity in acute viral hepatitis.
Mater Med Pol 1974; 6: 95–98.
26. Washio KW, Kusunoki Y, Murase T, et al. Xanthineoxidoreductase activity is correlated with insulin resistanceand subclinical inflammation in young humans. Metabolism
2017; 70: 51–56.
27. Martinez-Hervas S, Real JT, Ivorra C, et al. Increased plasma
xanthine oxidase activity is related to nuclear factor kappabeta activation and inflammatory markers in familial
combined hyperlipidemia. Nutr Metab Cardiovasc Dis 2010;
20: 734–739.

ª 2018 The Authors. Journal of Diabetes Investigation published by AASD and John Wiley & Sons Australia, Ltd

ORIGINAL ARTICLE
Plasma XO activities in T2DM and MetS

28. Kizaki H, Sakurada T. Simple micro-assay methods for
enzymes of purine metabolism. J Lab Clin Med 1977; 89:1135–1144.
29. Murase T, Nampei M, Oka M, et al. A highly sensitive assay
of human plasma xanthine oxidoreductase activity using
stable isotope-labeled xanthine and LC/TQMS. J Chromatogr
B Analyt Technol Biomed Life Sci 2016; 1039: 51–58.
30. Fujimura Y, Yamauchi Y, Murase T, et al. Relationshipbetween plasma xanthine oxidoreductase activity and left
ventricular ejection fraction and hypertrophy among cardiac
patients. PLoS ONE 2017; 12: e01826993.
31. Real JT, Martınez-Hervas S, Garcıa-Garcıa AB, et al.
Circulating mononuclear cells nuclear factor-kappa B
activity, plasma xanthine oxidase, and low grade
inflammatory markers in adult patients with familial
hypercholesterolaemia. Eur J Clin Invest 2010; 40: 89–94.
32. Salim S, Asghar M, Chugh G, et al. Oxidative stress: apotential recipe for anxiety, hypertension and insulin
resistance. Brain Res 2010; 1359: 178–185.
33. Cosic V, Antic S, Pesic M, et al. Monotherapy with
metformin: does it improve hypoxia in type 2 diabetic
patients? Clin Chem Lab Med 2001; 39: 818–821.

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