論文ID: CJ-18-0082
Background: Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the formation of uric acid from hypoxanthine and xanthine, leading to an increase in superoxide and reactive oxygen species. Activation of XOR promotes oxidative stress-related tissue injury. We investigated the associations between metabolic parameters and plasma XOR activity measured by a sensitive and accurate assay using a combination of liquid chromatography and triple quadrupole mass spectrometry to detect [13C2,15N2]-uric acid using [13C2,15N2]-xanthine as a substrate.
Methods and Results: A total of 627 Japanese subjects (M/F, 292/335) from the Tanno-Sobetsu Study, a population-based cohort, were recruited. Plasma XOR activity was significantly higher in males than in females, and habitual smoking was associated with elevation of activity. Plasma XOR activity was positively correlated with body mass index (BMI; r=0.323, P<0.001), waist circumference, blood pressure, and levels of liver enzymes including alanine transaminase (r=0.694, P<0.001), uric acid (r=0.249, P<0.001), triglycerides (r=0.312, P<0.001), hemoglobin A1c, fasting glucose, insulin and HOMA-R (r=0.238, P<0.001) as a marker of insulin resistance and was negatively correlated with high-density lipoprotein cholesterol level. On stepwise and multivariate regression analyses, BMI, smoking and levels of alanine transaminase, uric acid, triglycerides and HOMA-R were independent predictors of plasma XOR activity after adjustment for age and gender.
Conclusions: Plasma XOR activity is a novel biomarker of metabolic disorders in a general population.
Uric acid is the end product of purine metabolism in higher primates, including humans.1 Hyperuricemia is closely associated with visceral fat accumulation2,3 and various metabolic disorders conceptualized as metabolic syndrome, such as glucose intolerance, elevated blood pressure, dyslipidemia and atherosclerotic cardiovascular disease.4 Therefore, elevation of uric acid has been thought to be a possible marker of metabolic and cardiovascular diseases. It has not, however, necessarily been proven that the risk of cardiovascular events can be decreased by reduction of uric acid.5–7
Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid in the purine metabolism pathway.8 In mammals, XOR can convert 2 different forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO). XDH reduces NAD+ to NADH, whereas XO consumes oxygen to produce superoxide. XOR activity implies the total activity of both forms.9 XOR exists as the XDH form in several tissues, including the liver, intestine and other organs, and leaks into the blood and consequently converts to the XO form.10,11 Since activation of XOR promotes a resultant increase in superoxide,9 XOR is recognized as a significant source of reactive oxygen species, contributing to the development of oxidative stress-related tissue injury.12–14 It has, however, been difficult to accurately measure plasma XOR activity in humans because the activity is much lower in humans than in animals.15 Therefore, the relationship between plasma XOR activity and metabolic phenotype in humans remains to be elucidated.
Recently, a novel, sensitive and accurate assay for plasma XOR activity in humans has been established using a combination of liquid chromatography and triple quadrupole mass spectrometry (LC/TQMS) to detect [13C2,15N2]-uric acid using [13C2,15N2]-xanthine as a substrate.16 In the present study, we investigated the associations between plasma XOR activity and metabolic parameters in a general population.
The Tanno-Sobetsu Study involves a population-based cohort design in 2 rural towns, Tanno and Sobetsu, in Hokkaido, the northernmost island of Japan. Citizens aged ≥20 years in the towns were eligible for this cohort study. A total of 627 Japanese subjects (M/F, 292/335; mean age, 65±15 years) of Sobetsu Town who underwent annual examinations in 2016 were recruited into the present study. This study conformed to the principles outlined in the Declaration of Helsinki and was performed with the approval of the Ethics Committee of Sapporo Medical University. Written informed consent was received from all of the subjects.
Medical check ups were performed between 06:00 hours and 09:00 hours after overnight fast. After measuring anthropometric parameters, blood pressure was measured twice consecutively on the upper arm using an automated sphygmomanometer (HEM-907, Omron Co., Kyoto, Japan) with subjects in a seated resting position, and average blood pressure was used for analysis. Body mass index (BMI) was calculated as body weight (in kilograms) divided by the square of body height (in meters). Peripheral venous blood samples were obtained after physical examination for complete blood count and biochemistry. Samples of the serum and plasma were analyzed immediately or stored at −80℃ until biochemistry analysis.
MeasurementsPlasma glucose was measured using the glucose oxidase method. Fasting plasma insulin was measured by a chemiluminescent enzyme immunoassay. Hemoglobin A1c (HbA1c) was determined by a latex coagulation method and is expressed using the National Glycohemoglobin Standardization Program (NGSP) scale. Creatinine, blood urea nitrogen (BUN), uric acid, aspartate transaminase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γGTP) and lipid profiles, including total cholesterol, high-density lipoprotein cholesterol (HDL-C) and triglycerides, were measured using enzymatic methods. Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation. Brain natriuretic peptide (BNP) was measured using an assay kit (Shionogi & Co., Osaka, Japan). Homeostasis model assessment of insulin resistance (HOMA-R), an index of insulin resistance, was calculated using the previously reported formula: HOMA-R=insulin (μU/mL)×glucose (mg/dL)/405. As an index of renal function, estimated glomerular filtration rate (eGFR) was calculated using an equation for Japanese subjects:17 eGFR (mL/min/1.73 m2)=194×creatinine(−1.094)×age(−0.287)×0.739 (if female).
Plasma XOR ActivityThe modified assay protocol for plasma XOR activity in humans, which was established on the basis of assays for XOR activity in mice,18,19 was performed as previously reported.16 In brief, 100 μL of each plasma sample was purified by removing small molecules, including hypoxanthine, xanthine and uric acid, using a Sephadex G25 column and was mixed with 16 µmol/L [13C2,15N2]-xanthine as a substrate, 16 µmol/L NAD+ and 1 µmol/L [13C3, 15N3]-uric acid as an internal standard in 250 µL Tris buffer (pH 8.5). Each of the mixtures was incubated at 37℃ for 90 min, mixed with 500 μL methanol, and centrifuged at 2,000×g for 15 min at 4℃. The supernatants were transferred to new tubes and dried using a centrifugal evaporator. The residues were reconstituted with 150 μL distilled water and filtered through an ultrafiltration membrane before LC/TQMS using a Nano Space SI-2 LC system (Shiseido, Ltd., Tokyo, Japan) and a TSQ-Quantum triple quadrupole mass spectrometer (TQMS, Thermo Fisher Scientific, Bremen, Germany) equipped with an ESI interface. The amount of [13C2,15N2]-uric acid produced was quantified using the calibration curve, and XOR activity is expressed as [13C2,15N2]-uric acid in pmol/h/mL plasma. The lower detection limit was 6.67 pmol/h/mL plasma, and the intra- and inter-assay coefficients of variation of pooled human plasma XOR activity were 6.5% and 9.1%, respectively.16
Statistical AnalysisNumeric variables are expressed as mean±SD for normal distribution or median (IQR) for skewed variables. The distribution of each parameter was tested for normality using Shapiro-Wilk W-test, and non-normally distributed parameters were logarithmically transformed for regression analysis. Comparison between 2 groups was done with Student’s t-test for parametric parameters and Mann-Whitney U-test for non-parametric parameters. Intergroup differences in proportions in demographic parameters were examined with chi-squared test. The correlation between 2 variables was evaluated using Pearson’s correlation coefficient. Stepwise and subsequent multivariate regression analyses were performed to identify independent determinants of plasma XOR activity using age, gender and the variables with a significant after consideration of multicollinearity, and with the t-ratio calculated as the ratio of the unstandardized regression coefficient and SE of the unstandardized regression coefficient, the standardized regression coefficient (β), the percentage of variance in the object variables that the selected independent predictors explained (R2), and the Akaike information criterion (AIC). Of the candidate models, the best-fit model using AIC for each dependent variable was selected. P<0.05 was considered statistically significant. All data were analyzed using JMP 9 for Macintosh (SAS Institute, Cary, NC, USA).
Characteristics of the 627 recruited subjects (M/F, 292/335) are listed in Table 1. Hypertension (use of antihypertensive drugs, systolic blood pressure [SBP] ≥140 mmHg or diastolic blood pressure [DBP] ≥90 mmHg), diabetes mellitus (use of anti-diabetic drugs or a combination of HbA1c ≥6.5% and fasting glucose ≥126 mg/dL), dyslipidemia (use of anti-dyslipidemic drugs, LDL-C ≥140 mg/dL, HDL-C <40 mg/dL or triglycerides ≥150 mg/dL) and hyperuricemia (use of anti-hyperuricemic drugs or uric acid >7 mg/dL) were identified in 358, 68, 336, and 78 subjects, respectively. Male subjects had significantly larger BMI and waist circumference (WC), a significantly higher prevalence of current smoking and drinking, and higher DBP, AST, ALT, γGTP, BUN, creatinine, eGFR, uric acid, triglycerides, fasting glucose and plasma XOR activity (Figure 1A) and lower total cholesterol, LDL-C, HDL-C and BNP than did female subjects. No significant difference in age, SBP, insulin, HOMA-R or HbA1c was found between the male and female subjects.
| Total (n=627) |
Male (n=292) |
Female (n=335) |
P-value | |
|---|---|---|---|---|
| Age (years) | 65±15 | 64±16 | 65±15 | 0.460 |
| BMI (kg/m2) | 23.5±3.8 | 24.0±3.6 | 23.0±3.8 | 0.001 |
| WC (cm) | 85.6±10.9 | 86.9±10.5 | 84.5±11.2 | 0.006 |
| SBP (mmHg) | 135±22 | 136±19 | 134±23 | 0.316 |
| DBP (mmHg) | 76±11 | 77±11 | 75±12 | 0.044 |
| Pulse rate (beats/min) | 70±11 | 70±12 | 71±11 | 0.129 |
| Smoking habit | 105 (16.7) | 70 (24.0) | 35 (10.4) | <0.001 |
| Drinking habit | 261 (41.6) | 172 (58.9) | 89 (26.6) | <0.001 |
| Medication | ||||
| Antihypertensive drugs | 225 (35.9) | 101 (34.6) | 124 (37.0) | 0.559 |
| Anti-diabetic drugs | 59 (9.4) | 35 (12.0) | 24 (7.2) | 0.041 |
| Anti-dyslipidemic drugs | 123 (19.6) | 48 (16.4) | 75 (22.4) | 0.070 |
| Anti-hyperuricemic drugs | 9 (1.4) | 8 (2.7) | 1 (0.3) | 0.015 |
| Antiplatelet drugs | 39 (6.2) | 30 (10.3) | 9 (2.7) | <0.001 |
| Anticoagulant drugs | 14 (2.2) | 10 (3.4) | 4 (1.2) | 0.101 |
| Biochemistry | ||||
| AST (IU/L) | 22 (20–27) | 24 (20–29) | 22 (19–26) | <0.001 |
| ALT (IU/L) | 18 (14–24) | 21 (16–28) | 16 (13–21) | <0.001 |
| γGTP (IU/L) | 22 (16–33) | 28 (20–41) | 18 (14–25) | <0.001 |
| BUN (mg/dL) | 16±5 | 17±5 | 15±4 | <0.001 |
| Creatinine (mg/dL) | 0.8 (0.7–0.9) | 0.9 (0.8–1.0) | 0.7 (0.6–0.8) | <0.001 |
| eGFR (mL/min/1.73 m2) | 66.8±14.2 | 68.5±15.4 | 65.4±13.0 | 0.011 |
| Uric acid (mg/dL) | 5.4±1.3 | 6.0±1.2 | 4.8±1.1 | <0.001 |
| TC (mg/dL) | 209±36 | 199±34 | 217±36 | <0.001 |
| LDL-C (mg/dL) | 121±31 | 115±29 | 126±31 | <0.001 |
| HDL-C (mg/dL) | 62±18 | 57±16 | 67±18 | <0.001 |
| TG (mg/dL) | 92 (66–129) | 97 (69–147) | 88 (65–117) | 0.003 |
| FG (mg/dL) | 93 (87–103) | 95 (88–108) | 92 (85–99) | <0.001 |
| Insulin (μU/mL) | 8.3 (4.3–16.6) | 9.1 (4.2–17.6) | 8.0 (4.3–15.6) | 0.320 |
| HOMA-R | 1.96 (0.93–4.03) | 2.17 (0.96–4.39) | 1.82 (0.92–3.61) | 0.113 |
| HbA1c (%) | 5.5 (5.2–5.8) | 5.5 (5.2–5.9) | 5.5 (5.2–5.7) | 0.138 |
| BNP (pg/mL) | 17 (10–33) | 15 (8–28) | 19 (11–36) | 0.001 |
| XOR (pmol/h/mL plasma) | 36 (21–66) | 43 (24–86) | 32 (20–53) | 0.002 |
Data given as n (%), mean±SD or median (IQR). AST, aspartate transaminase; ALT, alanine transaminase; BMI, body mass index; BNP, brain natriuretic peptide; BUN, blood urea nitrogen; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; FG, fasting glucose; γGTP, γ-glutamyl transpeptidase; HDL-C, high-density lipoprotein cholesterol; HOMA-R, homeostasis model assessment of insulin resistance; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglycerides; WC, waist circumference; XOR, xanthine oxidoreductase.
Plasma xanthine oxidoreductase (XOR) activity according to (A) gender, (B) smoking status, (C) diabetes mellitus status, (D) dyslipidemia status, (E) hyperuricemia status and (F) anti-hyperuricemic drug treatment. *P<0.05.
Plasma XOR activity was significantly higher in smokers than in non-smokers (Figure 1B). There was no significant difference in plasma XOR activity according to alcohol drinking status or to hypertension status. The subjects with diabetes mellitus (Figure 1C), dyslipidemia (Figure 1D) or hyperuricemia (Figure 1E) had significantly higher plasma XOR activity than did those without. There was no significant difference in plasma XOR activity in hyperuricemic subjects according to anti-hyperuricemic drug treatment status (Figure 1F).
Plasma XOR Activity and XOR InhibitorsNine subjects had been treated with anti-hyperuricemic drugs (Table 2). They had not been treated with diuretics, which may cause hyperuricemia. The anti-hyperuricemic drugs included XOR inhibitors, allopurinol (n=6) and febuxostat (n=2), and a suppressor of uric acid reabsorption in the proximal renal tubule, benzbromarone (n=1). In subjects 1–6 (male), who were being treated with XOR inhibitors, uric acid was ≤7.0 mg/dL (the cut-off for hyperuricemia), and plasma XOR activity was lower than the median for male subjects (43 pmol/h/mL plasma). Plasma XOR activity in subject 7 (male), who was being treated with a non-XOR inhibitor, benzbromarone, was relatively high (53.2 pmol/h/mL plasma), and uric acid was 7.2 mg/dL. In subject 8 (female), who was being treated with an XOR inhibitor, allopurinol, uric acid was <7.0 mg/dL, but plasma XOR activity (138 pmol/h/mL plasma) was approximately 4.3-fold higher than the median for female subjects (32 pmol/h/mL plasma). In subject 9 (male), who was being treated with an XOR inhibitor, febuxostat, plasma XOR activity was 346 pmol/h/mL plasma, which was approximately 8-fold higher than the median, and uric acid was also high (9.6 mg/dL). Subjects 8 and 9, who had high plasma XOR activity, had high BMI, large WC, and liver dysfunction and were being treated with anti-diabetic drugs (Table 2).
| Subject No. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|---|---|---|---|---|---|---|---|---|---|
| Age (years) | 81 | 77 | 74 | 74 | 74 | 73 | 53 | 75 | 52 |
| Gender | Male | Male | Male | Male | Male | Male | Male | Female | Male |
| XOR inhibitors | Allo | Allo | Febu | Allo | Allo | Allo | – | Allo | Febu |
| Non-XOR inhibitors | – | – | – | – | – | – | Ben | – | – |
| XOR (pmol/h/mL plasma) | 10.5 | 11.6 | 13.8 | 18.4 | 28.5 | 39.0 | 53.2 | 138 | 346 |
| Uric acid (mg/dL) | 5.7 | 7.0 | 5.1 | 4.9 | 6.2 | 5.2 | 7.2 | 6.1 | 9.6 |
| BMI (kg/m2) | 27.2 | 26.5 | 21.1 | 27.5 | 21.3 | 28.8 | 29.1 | 33.7 | 37.1 |
| WC (cm) | 89.0 | 97.5 | 77.0 | 99.0 | 86.0 | 103.6 | 98.0 | 119.0 | 115.5 |
| AST (IU/L) | 21 | 23 | 33 | 17 | 23 | 27 | 20 | 48 | 31 |
| ALT (IU/L) | 15 | 23 | 40 | 9 | 13 | 24 | 25 | 45 | 47 |
| γGTP (IU/L) | 28 | 27 | 60 | 13 | 46 | 65 | 44 | 28 | 32 |
| eGFR (mL/min/1.73 m2) | 58.8 | 38.6 | 47.1 | 47.1 | 76.2 | 49.5 | 61.4 | 53.0 | 61.7 |
| HDL-C (mg/dL) | 49 | 41 | 40 | 35 | 68 | 40 | 48 | 43 | 50 |
| TG (mg/dL) | 122 | 139 | 158 | 139 | 60 | 265 | 145 | 108 | 116 |
| FG (mg/dL) | 95 | 90 | 95 | 89 | 96 | 154 | 91 | 116 | 125 |
| Insulin (μU/mL) | 1.6 | 2.2 | 2.6 | 0.3 | 2.3 | 2.2 | 1.7 | 3.5 | 3.3 |
| HOMA-R | 1.17 | 2.01 | 3.08 | 0.29 | 2.29 | 3.53 | 1.26 | 9.08 | 8.15 |
| HbA1c (%) | 5.6 | 5.4 | 5.5 | 5.5 | 5.4 | 7.0 | 5.7 | 6.3 | 6.6 |
| Diuretics | – | – | – | – | – | – | – | – | – |
| Anti-HT drugs | + | + | + | + | + | + | − | + | + |
| Anti-DM drugs | − | − | − | − | − | + | − | + | + |
| Anti-DL drugs | − | − | + | − | − | − | − | − | − |
| Smoking habit | − | − | − | − | − | − | + | − | − |
Allo, allopurinol; BEN, benzbromarone; DL, dyslipidemia; DM, diabetes mellitus; Febu, febuxostat; HT, hypertension. Other abbreviations as in Table 1.
Plasma XOR activity was positively correlated with BMI (Figure 2A), WC, DBP and AST (Figure 2B), ALT (Figure 2C), γGTP, eGFR, uric acid (Figure 2D), triglycerides (Figure 2E), fasting glucose, insulin, HOMA-R (Figure 2F) and HbA1c, and was negatively correlated with HDL-C (Table 3). Similar correlations between the parameters were observed when male and female subjects were analyzed separately.
(A) Body mass index (BMI), (B) logarithmically transformed (log) aspartate transaminase (AST), (C) log alanine aminotransferase (ALT), (D) uric acid, (E) log triglycerides and (F) log homeostasis model assessment of insulin resistance (HOMA-R) vs. log plasma xanthine oxidoreductase (XOR) activity in (○, - - -) men (n=292) and (●, –) women (n=335); total group, n=627 (M/F, 292/335).
| Total (n=627) | Male (n=292) | Female (n=335) | ||||
|---|---|---|---|---|---|---|
| r | P-value | r | P-value | r | P-value | |
| Age | 0.001 | 0.974 | −0.094 | 0.109 | 0.121 | 0.027 |
| BMI | 0.323 | <0.001 | 0.331 | <0.001 | 0.285 | <0.001 |
| WC | 0.286 | <0.001 | 0.293 | <0.001 | 0.256 | <0.001 |
| SBP | 0.069 | 0.083 | 0.004 | 0.952 | 0.119 | 0.030 |
| DBP | 0.115 | 0.004 | 0.145 | 0.013 | 0.065 | 0.240 |
| Pulse rate | −0.003 | 0.941 | 0.024 | 0.694 | −0.001 | 0.877 |
| Log AST | 0.586 | <0.001 | 0.590 | <0.001 | 0.550 | <0.001 |
| Log ALT | 0.694 | <0.001 | 0.709 | <0.001 | 0.642 | <0.001 |
| Log γGTP | 0.432 | <0.001 | 0.362 | <0.001 | 0.444 | <0.001 |
| BUN | −0.037 | 0.360 | −0.173 | 0.003 | 0.068 | 0.213 |
| Log creatinine | 0.009 | 0.818 | −0.158 | 0.007 | −0.058 | 0.288 |
| eGFR | 0.096 | 0.016 | 0.159 | 0.006 | −0.017 | 0.753 |
| Uric acid | 0.249 | <0.001 | 0.208 | <0.001 | 0.163 | 0.003 |
| Total cholesterol | 0.030 | 0.451 | 0.061 | 0.298 | 0.101 | 0.066 |
| LDL-C | 0.037 | 0.351 | 0.044 | 0.457 | 0.106 | 0.052 |
| HDL-C | −0.191 | <0.001 | −0.169 | 0.004 | −0.129 | 0.018 |
| Log TG | 0.312 | <0.001 | 0.284 | <0.001 | 0.305 | <0.001 |
| Log FG | 0.203 | <0.001 | 0.169 | 0.004 | 0.189 | 0.001 |
| Log insulin | 0.214 | <0.001 | 0.235 | <0.001 | 0.185 | 0.001 |
| Log HOMA-R | 0.238 | <0.001 | 0.251 | <0.001 | 0.207 | <0.001 |
| Log HbA1c | 0.199 | <0.001 | 0.167 | 0.004 | 0.213 | <0.001 |
| Log BNP | −0.052 | 0.194 | −0.019 | 0.743 | −0.043 | 0.438 |
Abbreviations as in Table 1.
On stepwise and subsequent multivariate regression analyses for plasma XOR activity using age, gender, BMI, DBP, smoking, ALT, uric acid, eGFR, HDL-C, triglycerides, HOMA-R and HbA1c as possible determinants, BMI, smoking, ALT, uric acid, triglycerides and HOMA-R were independently associated with plasma XOR activity after adjustment for age and gender, explaining a total of 50.7% of the variance in this measure (R2=0.507; Table 4).
| Regression coefficient |
SE | Standardized regression coefficient (β) |
t | P-value | |
|---|---|---|---|---|---|
| Age | 0.005 | 0.002 | 0.082 | 2.61 | 0.009 |
| Gender (Male) | −0.052 | 0.029 | −0.006 | −1.76 | 0.078 |
| BMI | 0.018 | 0.007 | 0.079 | 2.41 | 0.016 |
| Smoking | 0.088 | 0.036 | 0.078 | 2.43 | 0.015 |
| Log ALT | 1.132 | 0.063 | 0.608 | 18.10 | <0.001 |
| Uric acid | 0.057 | 0.022 | 0.088 | 2.53 | 0.012 |
| Log TG | 0.155 | 0.052 | 0.095 | 2.96 | 0.003 |
| Log HOMA-R | 0.062 | 0.024 | 0.079 | 2.57 | 0.010 |
R2=0.507. Abbreviations as in Table 1.
The present study demonstrated that plasma XOR activity was independently associated with BMI, smoking, and levels of ALT, uric acid, triglycerides and HOMA-R as an index of insulin resistance in a general population, suggesting that it may be a novel biomarker of metabolic disorders. It has been difficult to measure plasma XOR activity in humans because the activity is extremely low. Therefore, there have been only a few reports on the amount and activity of XOR in humans.20–22 In the present study, we measured plasma XOR activity using a novel, sensitive and accurate assay, which has recently been established using LC/TQMS to detect [13C2,15N2]-uric acid with [13C2,15N2]-xanthine as a substrate.16 A previous study using a small number (n=29) of young volunteers (mean age, 25.9 years) showed that plasma XOR activity assessed using the same assay as that in the present study was positively correlated with BMI and insulin resistance.23 In the present study, we revealed independent associations between plasma XOR activity and metabolic parameters using a large number of subjects.
XOR is expressed as the XDH form in tissues, with a large amount in the liver, and it leaks into the blood and consequently converts to the XO form.9,10 Since XO is shed by the liver without non-specific membrane damage into plasma and is partially bound to vascular endothelial cells,12 the main source of plasma XOR activity might be XO released from the liver, being consistent with strong correlations between plasma XOR activity and liver enzymes, including ALT (r=0.694, P<0.001), AST (r=0.586, P<0.001) and γGTP (r=0.432, P<0.001), found in the present study. Further investigations are needed to determine whether the increase in XOR activity affects release of XO from the liver and whether the production of XO increases metabolic disorders.
XOR activity is suggested to be one of the links between cigarette smoking and cardiovascular disease.24 Tobacco smoke condensate or extract has been reported to increase the expression and activity of XOR in cultured pulmonary endothelial cells, resulting in oxidative stress and cell apoptosis.25–27 Pretreatment of endothelial cells with an XOR inhibitor, allopurinol or febuxostat, decreased cigarette smoke-mediated DNA damage and cell apoptosis.27 In rodents, cigarette smoke exposure also increased XOR activity, resulting in smoke-induced vascular damage.26,27 Furthermore, allopurinol, an XOR inhibitor, reversed smoking-induced endothelial dysfunction and alteration of vessel resistance in humans.28,29 In the present study, current smoking was independently associated with plasma XOR activity.
Adipose tissue abundantly expresses XOR and can produce uric acid through XOR, which is enhanced in obesity-related insulin resistance.30 Insulin resistance impairs glycolysis and activates the pentose phosphate pathway, leading to the promotion of a hepatic de novo purine synthetic pathway, thereby triggering hyperuricemia.31 Fatty acid synthesis is increased in obese adipose tissue,32 and the increase in fatty acid synthesis is closely associated with de novo purine synthesis through activation of the pentose phosphate pathway.31 Furthermore, XOR-null mice have low plasma fatty acid and lipid accumulation in mammary epithelium and renal tubules,33–35 suggesting the possible involvement of XOR in lipid homeostasis. This suggests possible associations of plasma XOR activity with obesity, hyperuricemia, dyslipidemia and insulin resistance, which was confirmed in the present study by showing independent associations of plasma XOR activity with metabolic parameters (Table 4) and high plasma XOR activities in subjects with diabetes mellitus, dyslipidemia and hyperuricemia (Figure 1C–E).
Lowering of uric acid level has been shown to improve cardiovascular and mortality outcomes.36–39 Reduction of uric acid, however, does not necessarily decrease cardiovascular events.5–7 In contrast, XOR activity in tissue and/or plasma is associated with the incidence of heart failure, cardiovascular events and renal dysfunction.12,21,40 In animal models, inhibition of XOR activity by allopurinol, febuxostat or topiroxostat improved cardiovascular and renal outcomes, by reducing superoxide-induced tissue injury.41,42 Furthermore, high-dose allopurinol improves endothelial function by profoundly reducing vascular oxidative stress but not by lowering uric acid in humans.43 In the present study, despite treatment with XOR inhibitors, 2 of the 9 subjects with hyperuricemia (subjects 8 and 9) still had high plasma XOR activity, and those subjects had high BMI, large WC, and liver dysfunction and were being treated for diabetes mellitus (Table 2), indicating that they might be resistant to an XOR inhibitor. Furthermore, 1 subject (subject 8) had uric acid <7.0 mg/dL (cut-off for hyperuricemia), but had still high plasma XOR activity, indicating a sufficient effect of the XOR inhibitor for lowering uric acid but not for lowering plasma XOR activity. Adequate inhibition of plasma XOR activity, not just lowering uric acid level, might be a novel therapeutic strategy for metabolic and cardiovascular diseases.
Study LimitationsThe present study has several limitations. First, the design was cross-sectional, which means that a causal relationship cannot be proved between plasma XOR activity and correlated biomarkers. A longitudinal study and interventional study are needed to clarify the underlying relationship between plasma XOR activity and metabolic parameters. Second, because the recruited subjects were Japanese only, it is unclear whether the present findings can be generalized to other ethnicities. Third, plasma XOR activity measurement is not standardized across laboratories. Therefore, plasma XOR activity measured in the present study is not directly comparable to that measured in another laboratory using a different method. Fourth, the time after administration of drugs, especially for XOR inhibitors, may affect plasma XOR activity level, although venous blood samples were obtained in the early morning after overnight fast. Last, since pretreatment of plasma samples using a Sephadex G25 for removing small molecules also removes the drug molecules, the present protocol may have affected the values of plasma XOR activity in subjects treated with XOR inhibitors. For interventional studies using XOR inhibitors, this issue needs to be clarified.
Plasma XOR activity is independently associated with BMI, smoking and levels of transaminase, uric acid, triglycerides and insulin resistance index in a general population, suggesting that it is a novel biomarker of metabolic disorders. Measurement of XOR activity may help to identify patients with a high risk of metabolic and cardiovascular diseases. Further study is needed to elucidate the mechanism underlying the link between plasma XOR activity and tissue injury.
M.F. has been supported by grants from JSPS KAKENHI.
T. Murase and T.N. received a salary from Sanwa Kagaku Kenkyusho Co., Ltd. to develop the assay protocol for plasma XOR activity and measure its activity. This does not affect the sharing of data or materials. The other authors declare no conflict of interest.
