activity in female subjects with low levels of uric acid

©The Japan Endocrine Society

2018, 65 (11), 1083-1092

Original

Unexpected high plasma xanthine oxidoreductaseactivity in female subjects with low levels of uric acidMasato Furuhashi1), Kazuma Mori1), Marenao Tanaka1), Takuto Maeda2), Megumi Matsumoto1),Takayo Murase3), Takashi Nakamura3), Masayuki Koyama1), 4), Norihito Moniwa1), Hirofumi Ohnishi1), 4),Shigeyuki Saitoh1), 5), Kazuaki Shimamoto6) and Tetsuji Miura1)

1) Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8543,Japan

2) Department of Nephrology, Teine Keijinkai Hospital, Sapporo 006-8555, Japan3) Sanwa Kagaku Kenkyusho Co., Ltd., Inabe 511-0406, Japan4) Department of Public Health, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan5) Department of Nursing, Division of Medical and Behavioral Subjects, Sapporo Medical University School of Health Sciences,

Sapporo 060-8543, Japan6) Japan Health Care College, Sapporo 004-0839, Japan

Abstract. Hypouricemia is a high-risk factor of exercise-induced acute kidney injury (EIAKI) probably through a lack of anantioxidant effect of uric acid. Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the formation of uric acid fromhypoxanthine and xanthine, leading to an increase in superoxide and reactive oxygen species. Activation of XOR has beenproposed to promote oxidative stress-related tissue injury. We measured plasma XOR activity by a sensitive and accurateassay using a combination of liquid chromatography and triple quadrupole mass spectrometry in subjects with relatively lowlevels of uric acid (≤4.0 mg/dL) who were recruited from 627 subjects (male/female: 292/335) in the Tanno-Sobetsu Study, apopulation-based cohort. The numbers of subjects with uric acid ≤4.0 mg/dL, ≤3.0 mg/dL and ≤2.0 mg/dL were 72 (11.5%,male/female: 5/67), 13 (2.1%, all females) and 2 (0.3%, both females), respectively. Plasma XOR activities in 5 male subjectswere below the median value of the 292 male subjects. In 12 (17.9%) of the 67 female subjects with uric acid ≤4.0 mg/dL,plasma XOR activities were above the upper quartile value of the 335 female subjects. Eleven of the 12 female subjects withhigh plasma XOR activity and a low uric acid level had liver dysfunction and/or insulin resistance. In conclusion, unexpectedhigh plasma XOR activities were found in some female subjects with relatively low levels of uric acid. Measurement ofplasma XOR activity may help to identify hypouricemic patients with a high risk for EIAKI.

Key words: Xanthine oxidoreductase, Renal hypouricemia, Liver dysfunction, Insulin resistance, Exercise-induced acutekidney injury

URIC ACID is the end product of purine metabolism inhumans [1]. Hyperuricemia is closely associated withvarious metabolic disorders and atherosclerotic cardio‐vascular diseases [2]. Therefore, elevation of uric acidlevel has been thought to be a surrogate marker of meta‐bolic syndrome and cardiovascular diseases. On the other

Submitted Mar. 28, 2018; Accepted Jul. 4, 2018 as EJ18-0127Released online in J-STAGE as advance publication Aug. 2, 2018Correspondence to: Masato Furuhashi, M.D., Ph.D., Department ofCardiovascular, Renal and Metabolic Medicine, Sapporo MedicalUniversity School of Medicine, S-1, W-16, Chuo-ku, Sapporo060-8543, Japan.E-mail: [email protected]

hand, hypouricemia per se is mostly asymptomatic, butthe condition is well known to be a high risk for urolith‐iasis and exercise-induced acute kidney injury (EIAKI)[3]. Previous studies demonstrated that the prevalence ofhypouricemia (uric acid ≤2.0 mg/dL) by unknown etiol‐ogy was about 0.2~0.6%, being larger in female subjectsthan in male subjects, in the general population [4-6].

Renal hypouricemia results from an isolated renal tubu‐lar defect in reabsorption and/or secretion of uric acidthat is genetically determined in an autosomal recessiveinheritance pattern [7]. The main etiology of renal hypo‐uricemia is a gene mutation of urate transporters includ‐ing solute carrier family 22, member 12 (SLC22A12),

also known as urate transporter 1 (URAT1), and solutecarrier family 2, member 9 (SLC2A9), also known asglucose transporter 9 (GLUT9) or voltage-driven urateefflux transporter (URATv1) [8-11]. The incidence ofrenal hypouricemia has been reported to range from0.12% to 0.20% [12-14].

In 71 patients (male/female: 43/28) with renal hypouri‐cemia based on genetic testing, it was reported that acuterenal failure and urolithiasis as complications occurred in15 patients (21.1%, male/female: 12/3) and 6 patients(8.5%, male/female: 5/1), respectively [15]. It has beenproposed that hypouricemia causes EIAKI through oxi‐dative stress-induced spasm of the renal artery, since uricacid acts as an antioxidant to protect endothelial function[16-18]. Interestingly, unlike renal hypouricemia causedby hyperexcretion of uric acid, hypouricemia caused bylow production of uric acid in human xanthine oxidore‐ductase (XOR) deficiency (hereditary xanthinuria) hasnot been reported to cause EIAKI [14, 19, 20], suggest‐ing that XOR activity may play a role in the developmentof EIAKI in hypouricemia.

XOR is an enzyme that catalyzes the formation of uricacid from hypoxanthine and xanthine, leading to anincrease in superoxide and reactive oxygen species [21].Therefore, activation of XOR contributes to the develop‐ment of oxidative stress-related tissue injury [22-24].However, it has been difficult to accurately measureplasma XOR activity in humans since the activity ismuch lower in humans than in animals [25]. Recently, anovel, sensitive and accurate assay for plasma XORactivity in humans has been established using a combina‐tion of liquid chromatography and triple quadrupolemass spectrometry (LC/TQMS) to detect [13C2, 15N2]-uric acid using [13C2, 15N2]-xanthine as a substrate [26].Plasma XOR activity has been reported to be associatedwith obesity, smoking, liver dysfunction, hyperuricemia,dyslipidemia, insulin resistance and adiponectin [27-29].

We hypothesized that plasma XOR activity plays acrucial role in the induction of complications in patientswith renal hypouricemia. However, it is very difficult torecruit patients with renal hypouricemia and to prove ourhypothesis, since the number of patients who have renalhypouricemia with and without complications is verysmall. Therefore, in the present study, we investigatedplasma XOR activity in subjects with relatively low lev‐els of serum uric acid in a general population as the firststep for proving our hypothesis.

Materials and Methods

Study populationIn the Tanno-Sobetsu Study, a study with a population-

based cohort design in two rural towns, Tanno andSobetsu, in Hokkaido, the northernmost island of Japan,a total of 627 Japanese subjects (male/female: 292/335,mean age: 65 ± 15 years) were recruited from residentsof Sobetsu Town in 2016. The population was the sameas that in our previous study for investigating plasmaXOR activity [28]. Hypouricemia is conventionallydefined as a serum uric acid concentration of ≤2 mg/dL[4-6, 12-14]. However, it has been reported that levels ofuric acid are more than 3 mg/dL in some patients withrenal hypouricemia who have heterozygote mutations ofSLC22A12/URAT1 or SLC2A9/GLUT9/URATv1 [15,30]. Therefore, subjects with relatively low levels of uricacid (≤4.0 mg/dL) were enrolled in the present study.Subjects diagnosed with diabetes mellitus (HbA1c ≥6.5%and fasting glucose ≥126 mg/dL) and subjects beingtreated with antidiabetic drugs, XOR inhibitors includingallopurinol, febuxostat and topiroxostat, and some angio‐tensin II receptor blockers (losartan and irbesartan) wereexcluded because of possible reduction of uric acid level.This study conformed to the principles outlined in theDeclaration of Helsinki and was performed with theapproval of the Ethical Committee of Sapporo MedicalUniversity. Written informed consent was received fromall of the study subjects.

Medical checkups were performed between 06:00 hand 09:00 h after an overnight fast. After measuringanthropometric parameters, blood pressure was measuredtwice consecutively on the upper arm using an automatedsphygmomanometer (HEM-907, Omron Co., Kyoto,Japan) with subjects in a seated resting position, andaverage blood pressure was used for analysis. Body massindex (BMI) was calculated as body weight (in kilo‐grams) divided by the square of body height (in meters).After physical examination, peripheral venous bloodsamples were obtained from the subjects for completeblood count and biochemical analyses. Samples of serumand plasma were analyzed immediately or stored at–80°C until biochemical analyses.

MeasurementsPlasma glucose was determined by the glucose oxidase

method. Fasting plasma insulin was measured by achemiluminescent enzyme immunoassay method.Hemoglobin A1c (HbA1c) was determined by a latex

1084 Furuhashi et al.

coagulation method and was expressed in NationalGlycohemoglobin Standardization Program (NGSP)scale. Creatinine, blood urea nitrogen (BUN), uric acid,aspartate transaminase (AST), alanine aminotransferase(ALT), γ-glutamyl transpeptidase (γGTP) and lipid pro‐files, including total cholesterol, HDL cholesterol andtriglycerides, were determined by enzymatic methods.LDL cholesterol level was calculated by the Friedewaldequation. Homeostasis model assessment of insulinresistance (HOMA-R), an index of insulin resistance,was calculated by the previously reported formula:HOMA-R = insulin (μU/mL) × glucose (mg/dL)/405. Asan index of renal function, estimated glomerular filtra‐tion rate (eGFR) was calculated by an equation forJapanese [31]: eGFR (mL/min/1.73 m2) = 194 × creati‐nine(–1.094) × age(–0.287) × 0.739 (if female).

Plasma XOR activityThe modified assay for plasma XOR activity in

humans, which was established on the basis of assays forXOR activity in mice [32, 33], was performed as previ‐ously reported [26, 28]. In brief, 100 μL of each plasmasample was purified by removing small molecules,including hypoxanthine, xanthine and uric acid, using aSephadex G25 column and was mixed with 16 μM [13C2,15N2]-xanthine as a substrate, 16 μM NAD+ and 1 μM[13C3, 15N3]-uric acid as an internal standard in 250 μLTris buffer (pH 8.5). Each of the mixtures was incubatedat 37°C for 90 min, mixed with 500 μL methanol, andcentrifuged at 2,000 × g for 15 min at 4°C. The super‐natants were transferred to new tubes and dried by a cen‐trifugal evaporator. The residues were reconstituted with150 μL of distilled water, filtered through an ultrafiltra‐tion membrane, and subjected to LC/TQMS using aNano Space SI-2 LC system (Shiseido, Ltd., Tokyo,Japan) and a TSQ-Quantum triple quadrupole mass spec‐trometer (TQMS, Thermo Fisher Scientific, Bremen,Germany) equipped with an ESI interface. The amountsof [13C2, 15N2]-uric acid produced were quantitated fromthe calibration curve, and XOR activities were expressedas [13C2, 15N2]-uric acid in pmol/h/mL plasma. The lowerdetection limit was 6.67 pmol/h/mL plasma, and intra-and inter-assay coefficients of variation of pooled humanplasma XOR activity were 6.5% and 9.1%, respectively[26].

Statistical analysisNumeric variables are expressed as means ± SD for

normal distributions or medians (interquartile ranges) for

skewed variables. The distribution of each parameter wastested for its normality using the Shapiro-Wilk W test.Comparison between two groups was done with Student’st test for parametric parameters and the Mann-Whitney Utest for nonparametric parameters. Intergroup differencesin percentages of demographic parameters were exam‐ined by the chi-square test. A p value of less than 0.05was considered statistically significant. All data wereanalyzed by using JMP 9 for Macintosh (SAS Institute,Cary, NC).

Results

Basal characteristics of the recruited subjectsUric acid levels in male subjects (n = 292) and female

subjects (n = 335) of the 627 subjects were 6.0 ± 1.2 and4.8 ± 1.1 mg/dL, respectively (Fig. 1A, B). The numberof subjects with a low level of uric acid (≤4.0 mg/dL) inthe 627 subjects was 89 (male/female: 10/79). Afterexclusion of subjects diagnosed with diabetes mellitus(n = 3) and subjects being treated with antidiabetic drugs(n = 11), XOR inhibitors (n = 0), losartan (n = 2) or irbe‐sartan (n = 1), 72 subjects (male/female: 5/67) wererecruited in the present study. Characteristics of therecruited subjects with low levels of uric acid are shownin Table 1. Mean age, BMI and waist circumference ofthe recruited subjects were 63 ± 16 years, 21.9 ± 3.0kg/m2 and 82.3 ± 10.4 cm, respectively. The numbers ofsubjects with smoking and drinking habits were 10(13.9%) and 16 (22.2%), respectively. Of the 72 subjects,20 (27.8%) and 11 (15.3%) subjects were taking antihy‐pertensive and antidyslipidemic drugs, respectively. Asignificantly larger percentage of male subjects had adrinking habit, and male subjects had a significantlyhigher creatinine level and significantly lower level ofinsulin. No significant differences in other parameterswere found between the male and female subjects. Thenumbers of subjects with uric acid ≤4.0 mg/dL, ≤3.0mg/dL and ≤2.0 mg/dL were 72 (11.5%, male/female:5/67), 13 (2.1%, all females) and 2 (0.3%, both females),respectively (Table 2).

Plasma XOR activities in the recruited subjects withlow levels of uric acid

In the 627 subjects (male/female: 292/335), plasmaXOR activities [median (interquartile ranges)] in themale and female subjects were 43.4 (24.4–86.3) and 32.3(19.8–53.4) pmol/h/mL plasma, respectively (Fig. 1C,D), as shown in our previous study [28]. Plasma XOR

XOR activity and hypouricemia 1085

Fig. 1 Plasma XOR activities in subjects with low levels of uric acid.A–D. Frequency distribution analyses of uric acid (mg/dL) (A, male; B, female) and plasma xanthine oxidoreductase (XOR)activity (pmol/h/mL plasma) (C, male; D, female) in the 627 recruited subjects (male/female: 292/335). E, F. Plasma XOR activitywas plotted against uric acid level in each subject with a relatively low level of uric acid (≤4.0 mg/dL) (E, male, n = 5; F, female, n= 67). The quartiles of plasma XOR activity in male (n = 292) and female (n = 335) Japanese subjects were from a previous report(Ref. 28). Q1, the first (lower) quartile of the activity; Q2, the second (median) quartile of the activity; Q3, the third (upper)quartile of the activity. Open triangles: male subjects with activity below of the Q3 value (n = 5), Open and closed circles: femalesubjects with activity below of the Q3 value (n = 55) and with activity of the Q3 value or more (n = 12), respectively.


activities in 5 male subjects were below the secondquartile (median) value (Q2, 43.4 pmol/h/mL plasma) ofthe 292 male subjects (Fig. 1E). In 12 (17.9%) of the 67female subjects with uric acid ≤4.0 mg/dL, plasma XORactivities were above the third (upper) quartile value(Q3, 53.4 pmol/h/mL plasma) of the 335 female subjects(Fig. 1F).

Unexpected high plasma XOR activity in somesubjects with low levels of uric acid

Characteristics of the 12 female subjects with a lowlevel of uric acid and high plasma XOR activity areshown in Table 3. Two of those subjects had a smokinghabit. Subject 6 had been treated with antihypertensiveand antidyslipidemic drugs. Subjects 1–3, 5 and 9 had

Table 1 Characteristics of the 72 recruited subjects with low level of uric acid (≤4.0 mg/dL)

Total Male Female p

n 72 5 67

Age (years) 63 ± 16 67 ± 13 62 ± 16 0.479

Body mass index (kg/m2) 21.9 ± 3.0 23.1 ± 2.8 21.8 ± 3.0 0.332

Waist circumference (cm) 82.3 ± 10.4 85.3 ± 11.7 82.1 ± 10.4 0.513

Systolic blood pressure (mmHg) 131 ± 23 139 ± 18 130 ± 10 0.375

Diastolic blood pressure (mmHg) 73 ± 9 74 ± 4 72 ± 10 0.776

Pulse rate (beats/min) 70 ± 9 67 ± 9 70 ± 10 0.436

Smoking habit 10 (13.9) 1 (20.0) 9 (13.4) 0.693

Drinking habit 16 (22.2) 4 (80.0) 12 (17.9) 0.002

Medications

Antihypertensive drugs 20 (27.8) 3 (60.0) 17 (25.4) 0.095

Antidyslipidemic drugs 11 (15.3) 0 (0) 11 (16.4) 0.325

Biochemical data

AST (IU/L) 21 (18–25) 23 (21–40) 21 (17–25) 0.131

ALT (IU/L) 15 (12–21) 22 (17–25) 15 (12–20) 0.058

γGTP (IU/L) 17 (13–24) 24 (19–28) 17 (13–24) 0.118

Blood urea nitrogen (mg/dL) 14 ± 3 17 ± 3 14 ± 3 0.053

Creatinine (mg/dL) 0.7 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 0.001

eGFR (mL/min/1.73 m2) 73 ± 14 73 ± 6 73 ± 14 0.950

Uric acid (mg/dL) 3.4 ± 0.5 3.5 ± 0.3 3.4 ± 0.5 0.645

Total cholesterol (mg/dL) 214 ± 33 191 ± 31 216 ± 33 0.107

LDL cholesterol (mg/dL) 125 ± 28 112 ± 27 126 ± 28 0.314

HDL cholesterol (mg/dL) 68 ± 15 57 ± 13 68 ± 15 0.117

Triglycerides (mg/dL) 80 (67–107) 89 (61–103) 80 (66–111) 0.991

Fasting glucose (mg/dL) 88 (83–95) 88 (54–95) 88 (83–95) 0.974

Insulin (μU/mL) 7.1 (3.1–15.6) 2.5 (1.1–7.4) 7.3 (3.5–15.8) 0.046

HOMA-R 1.42 (0.76–3.46) 0.55 (0.21–1.76) 1.44 (0.78–3.60) 0.051

HbA1c (%) 5.4 (5.1–5.6) 5.2 (5.1–5.4) 5.4 (5.2–5.6) 0.166

XOR (pmol/h/mL plasma) 23 (18–44) 19 (18–29) 25 (18–46) 0.347

Variables are expressed as number (%), means ± SD or medians (interquartile ranges).AST, aspartate transaminase; ALT, alanine transaminase; BNP, brain natriuretic peptide; eGFR, estimated glomerular filtration rate; γGTP,γ-glutamyl transpeptidase; HOMA-R, homeostasis model assessment of insulin resistance; XOR, xanthine oxidoreductase.


liver dysfunction defined as above a cut-off level ofAST, ALT or γGTP. In subjects 3–9, 11 and 12, HOMA-R levels were more than 2.00 as a cut-off level of insulinresistance. All of the subjects except for subject 10 hadliver dysfunction and/or insulin resistance.

Discussion

The present study revealed for the first time that 12(17.9%) of the 67 female subjects with a relatively low

level of uric acid had unexpected high plasma XORactivities. Eleven of the 12 female subjects had liver dys‐function and/or insulin resistance. Plasma XOR activityhas been reported to be independently associated withobesity, smoking, hyperuricemia, dyslipidemia, liverdysfunction, insulin resistance and adiponectin in a gen‐eral population [27-29]. XOR is abundantly expressed inthe liver and leaks into the blood without non-specificmembrane damage [21, 34]. Strong correlations betweenplasma XOR activity and liver enzymes, including AST

Table 2 Uric acid level and XOR activity in the 72 recruited subjects

All (n = 627) Male (n = 292) Female (n = 335)XOR (pmol/h/mL plasma)

Male Female

UA ≤4 mg/dL 72 (11.5) 5 67 18.8 (18.1–29.3) 25.1 (18.1–45.9)

UA ≤3 mg/dL 13 (2.1) 0 13 — 22.6 (14.2–39.7)

UA ≤2 mg/dL 2 (0.3) 0 2 — 22.1 (9.5–34.7)

Variables are expressed as number, number (%), or medians (interquartile ranges).

Table 3 Characteristics of the 12 subjects with low uric acid level and high plasma XOR activity

Subject No. 1 2 3 4 5 6 7 8 9 10 11 12

Age (years) 74 51 50 59 67 62 81 59 79 85 70 68

Gender F F F F F F F F F F F F

XOR (pmol/h/mL plasma) 197.0 143.0 113.0 97.5 95.1 76.4 76.3 72.1 71.7 66.4 56.5 54.2

Uric acid (mg/dL) 3.9 3.9 4.0 4.0 2.8 3.6 3.9 3.6 3.7 3.1 4.0 3.4

Body mass index (kg/m2) 21.7 22.4 24.9 23.9 22.3 24.3 25.6 22.3 30.5 22.9 22.7 19.3

Waist circumference (cm) 79.0 80.0 93.0 97.0 78.0 85.0 84.0 73.0 109.0 83.0 90.0 74.0

AST (IU/L) 50 38 42 28 24 25 22 21 25 29 22 29

ALT (IU/L) 39 49 62 26 22 25 15 23 25 22 21 26

γGTP (IU/L) 30 17 37 19 30 21 12 27 48 24 15 13

eGFR (mL/min/1.73m2) 74.2 67.5 68.9 92.9 87.7 63.8 81.3 99.3 78.7 83.7 60.7 63.1

HDL cholesterol (mg/dL) 60 72 70 70 36 62 57 72 62 62 53 96

Triglycerides (mg/dL) 96 84 189 115 199 103 81 54 105 79 168 59

Fasting glucose (mg/dL) 87 79 84 92 85 95 99 84 87 106 95 84

Insulin (μU/mL) 3.5 6.9 24.0 13.2 17.6 17.2 14.7 45.5 12.2 3.0 32.2 22.2

HOMA-R 0.76 1.35 4.98 3.01 3.69 4.03 3.60 9.45 2.63 0.79 7.55 4.60

HbA1c (%) 5.5 5.8 5.4 5.4 5.6 5.5 5.6 5.3 5.9 5.8 5.5 5.1

Antihypertensive drugs – – – – – + – – – – – –

Antidyslipidemic drugs – – – – – + – – – – – –

Habitual smoking – – + – + – – – – – – –

eGFR, estimated glomerular filtration rate; γGTP, γ-glutamyl transpeptidase; HOMA-R, homeostasis model assessment of insulinresistance; XOR, xanthine oxidoreductase.


(r = 0.586, p < 0.001), ALT (r = 0.694, p < 0.001) andγGTP (r = 0.432, p < 0.001), were found in our previousstudy [28]. It has also been reported that adipose tissueabundantly expresses XOR and can produce uric acidthrough XOR, which is enhanced in obesity-related insu‐lin resistance [35]. In the absence of hyperuricemia, liverdysfunction and insulin resistance may have caused theunexpected high plasma XOR activities in subjects witha low level of uric acid in the present study, though thepossibility that increased plasma XOR activity was aresult of low level of uric acid cannot be completelydenied.

Uric acid has a powerful antioxidant effect [36] andplays a protective role in the kidney. The decreased anti‐oxidant potential in renal hypouricemia may lead to kid‐ney injury caused by oxidative stress [16-18]. Oxidativeimbalance with increased oxidative stress after exerciseand decreased antioxidant capacity in patients with renalhypouricemia may cause EIAKI [18]. Renal perfusion inpatients with renal hypouricemia would be diminishedduring exercise, and reperfusion after exercise may leadto renal damage through ischemia-reperfusion injury.Unlike renal hypouricemia caused by hyperexcretion ofuric acid, hypouricemia caused by low production of uricacid in hereditary xanthinuria (XOR deficiency) has notbeen reported to cause EIAKI [14, 19, 20], suggestingthat XOR activity may play a role in the induction ofEIAKI in patients with hypouricemia. Measurement ofXOR activity may help to identify hypouricemic patientswith a high risk of EIAKI.

It was previously reported that pretreatment with allo‐purinol, an XOR inhibitor, could prevent the onset ofEIAKI in a subject with renal hypouricemia [37]. Poten‐tial mechanisms of prevention of EIAKI onset werethought to be protection of renal vasoconstriction follow‐ing depletion of free radical scavengers including uricacid and reduction of increased free radicals with ische‐mia reperfusion injury [30, 38, 39]. It has also been sug‐gested that high plasma XOR activity is a metabolicparameter that is superior to uric acid level, and adequateinhibition of plasma XOR activity, not just lowering uricacid level, would be a novel therapeutic strategy for met‐abolic and cardiovascular diseases [28]. Therefore, evenin hypouricemia, plasma XOR activity would be impor‐tant for the development of oxidative stress-mediateddiseases. To prove our hypothesis that plasma XORactivity plays a crucial role in the induction of EIAKI inpatients with renal hypouricemia, further study is neededto elucidate plasma XOR activities at rest and in exercise

conditions in subjects with renal hypouricemia.The prevalence of subjects with uric acid ≤2.0 mg/dL

in the present study was 0.3%, which was similar to theprevalence of hypouricemia (0.2–0.4%) in previous stud‐ies using Japanese subjects [4-6]. The number of subjectswith a relatively low level of uric acid was larger infemale subjects than in male subjects in the presentstudy. As a possible reason, female hormones, estrogenand progesterone, have been suggested to decrease uricacid level [40]. Since uric acid level is generally lower infemales than in males [28, 41], the prevalence of hypour‐icemia would be higher in females than that in males. Infact, the incidences of renal hypouricemia were reportedto be 0.16% and 0.23% in normal male and femaleadults, respectively [42]. It has also been reported that 3(0.24%) of 1,249 male subjects and 7 (0.56%) of 1,256female subjects had uric acid <2.0 mg/dL among healthyJapanese children [43].

Conversely, it has been reported that acute renal fail‐ure probably due to EIAKI in subjects with renal hypour‐icemia based on genetic testing occurred in 12 (27.9%)of 43 male patients and 3 (10.7%) of 28 female patients[15]. Male predominance of EIAKI seems to be unrelat‐ed to the incidence of renal hypouricemia. Strenuous andprolonged exercise causes a sequential increase in oxy‐gen free radical levels [44-46], linking to the pathogene‐sis of ischemic acute kidney injury [47]. Patients withrenal hypouricemia may be at risk of free radical-induceddamage to the kidney during exercise. Exercise alsocauses increased levels of several mediators includingnoradrenaline, angiotensin II, arginine vasopressin, endo‐toxin, endothelin, cytokines and leukotrienes [46], lead‐ing to facilitation of renal ischemia. One possible reasonfor the male predominance of EIAKI is the probability ofstrenuous and prolonged exercise performed by males. Ithas also been reported that estrogen has potent anti-oxidant properties and may influence the degree ofexercise-induced muscle damage and repair processes[48]. This may be another explanation for the genderdifference in the incidence of EIAKI. Measurement ofplasma XOR activity, especially during exercise toler‐ance test, may be useful to predict the onset of EIAKI inboth males and females with hypouricemia.

The present study has several limitations. First, sincethe present study was a cross-sectional design, it isunclear whether hypouricemia was transient or persis‐tent. It has been reported that hypouricemia was transientin 40% of outpatients because hypouricemia is a heterol‐ogous disease composed of renal hypouricemia, xanthi‐


nuria and secondary hypouricemia frequently causedeither by drug administration or systemic diseases [12].Differential diagnosis of hypouricemia was not com‐pletely performed in the present study, though subjectswith conditions that may affect serum uric acid levels,including subjects diagnosed with diabetes mellitus andsubjects treated with antidiabetic drugs, XOR inhibitors,and some angiotensin II receptor blockers (losartan andirbesartan), were excluded. Second, the number of sub‐jects enrolled in the present study was small. A longitu‐dinal study with larger numbers of subjects is necessaryfor confirming the prevalence and impact of high plasmaXOR activity in subjects with relatively low levels ofuric acid. Third, complications including urinary stonesand EIAKI were not investigated in the present study.Fourth, because the recruited subjects were only Japa‐nese people, it is unclear whether the present findingscan be generalized to other ethnicities. Lastly, plasmaXOR activity measurement is not standardized acrosslaboratories. Therefore, values of plasma XOR activitymeasured in the present study are not directly compara‐ble to those measured in another laboratory using a dif‐ferent method.

In conclusion, unexpected high plasma XOR activitiespossibly associated with liver dysfunction and insulinresistance were found in some female subjects with arelatively low level of uric acid in a general population.Measurement of XOR activity may help to identifyhypouricemic patients with a high risk of EIAKI. Furtherstudy is needed to elucidate the link between plasmaXOR activity and EIAKI in both male and female sub‐jects with hypouricemia.

Acknowledgements

M.F. was supported by grants from JSPS KAKENHI.

Disclosures

All of the authors except for T.Mu. and T.N. do nothave any relevant declarations. Sanwa Kagaku KenkyushoCo., Ltd. provided support in the form of salaries forT.Mu. and T.N., who developed the assay protocol ofplasma XOR activity and measured its activity. This doesnot alter our adherence about sharing data and materials.

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