Study design and participants.
This cross-sectional analysis was conducted as part of the Hyogo Sleep Cardio-Autonomic Atherosclerosis (HSCAA) study [49-51]. In summary, the HSCAA study is a single-center cohort study which aims to investigate the interrelationships among sleep disorders, autonomic neuropathy, metabolic diseases, and atherosclerotic diseases [49-51]. The HSCAA study included patients aged 20 years and older with one or more cardiovascular risk factors (obesity, smoking, presence of cardiovascular event history, hypertension, dyslipidemia, diabetes mellitus, chronic kidney disease) and being treated at the Hyogo College of Medicine Hospital.
Since we started XOR measurements from 2018 for the subjects who were registered or followed in the HSCAA study, this cross-sectional study included 310 patients, from January 2018 to July 2021, who consented abdominal CT examinations. In the end, 223 patients were analyzed in the present study after excluding 87 with alcoholic habits (>30 g/day for males and >20 g/day for females), autoimmune hepatitis, viral hepatitis, and under treatment with XOR inhibitors.
The HSCAA study has been approved by the Ethics Committee of Hyogo College of Medicine Hospital (Approval No. 2351). Written informed consent was obtained from all subjects and the study was conducted in full accordance with the Declaration of Helsinki. The present study protocol was approved by the Ethics Committee of Hyogo College of Medicine Hospital (Approval No. 3601) and performed with an opt-out option, as explained in instructions posted on the website of the hospital. All methods in our study were performed in accordance with the relevant guidelines and regulations.
Visceral fat area and subcutaneous fat area
CT was performed using SIEMENS SOMATOM Definition AS+ or SOMATOM Definition H (Siemens Healthcare GmbH, Erlangen, Germany) with 10 mm slices. We evaluated the visceral fat area (VFA), subcutaneous fat area (SFA), and waist circumference using Ziostation 2 (AMIN Ltd., Tokyo, Japan). The abdominal circumference (AC) was measured at the umbilical height.
Hepatic steatosis and liver fibrosis
The L/S ratio and HSI were used to evaluate hepatic steatosis. Hepatic and splenic attenuation values were measured on non-contrast-CT scans using four circular region-of-interest (ROI) cursors in the liver and two in the spleen. In the liver, four ROIs were located in each of the right lobe and the left lobe of the liver. All measurements were manually obtained in regions of uniform parenchymal attenuation, with care being taken to avoid vessels, artifacts, and other areas that might have spuriously increased or decreased measurements. Calculation of the L/S ratio was as follow: L/S ratio = (Average attenuation value of the liver) / (Average attenuation value of the spleen) [9-12]. HSI was calculated from ALT, AST, BMI, gender, and the presence of diabetes mellitus [13].
In addition, the NAFLD fibrosis score (NFS) and the Fibrosis-4 (Fib-4) index were calculated to predict the progression of liver fibrosis in patients with L/S ratio <1.1 [47, 48]. NFS was calculated from age, BMI, AST, ALT, the presence of glucose intolerance, platelet count, and albumin [47]. It has been reported that by applying the high cutoff score (NFS >0.676), the presence of advanced fibrosis could be diagnosed with high accuracy [47]. The FIB-4 index was calculated from age, AST, ALT, and platelet count. It has been reported that its cutoff value <1.45 can exclude hepatic fibrosis, and its cutoff value >3.25 can predict hepatic fibrosis [52].
Plasma XOR activity measurement
The assay protocol of XOR activity in humans was reported previously [26–28]. In brief, 100 μL of plasma samples (purified by Sephadex G25 resin) were mixed with a Tris buffer (pH 8.5) containing [13C2,15N2] xanthine as a substrate, NAD+, and [13C3,15N3] UA as an internal standard. The mixtures were incubated at 37°C for 90 min. Subsequently, the mixtures were mixed with 500 µL of methanol and centrifuged at 2,000 × g for 15 min at 4°C. The supernatants were transferred to new tubes and dried using a centrifugal evaporator. The residues were reconstituted with 150 μL of distilled water, filtered through an ultrafiltration membrane, and measured using LC/TQMS. LC/TQMS comprised a Nano Space SI-2 LC system (Shiseido Co., Ltd., Tokyo, Japan) and a TSQ Triple Quadrupole LC-MS system (ThermoFisher Scientific GmbH, Bremen, Germany) equipped with an ESI interface. Calibration standard samples of [13C2,15N2] UA were also measured, and the amounts of production were quantitated from the calibration curve. XOR activities were expressed in pmol/mL/h [26-28].
Other parameters
At the same time as that for the CT scan, blood samples were taken for AST, ALT, UA, fasting blood glucose, immunoreactive insulin, total cholesterol (T-Chol), high-density lipoprotein cholesterol (HDL-Chol), and TG. Serum UA levels were measured using the uricase/peroxidase technique with an autoanalyzer using UA (Pureauto S UA Sekisui Medical, Ltd., Tokyo, Japan). Height, weight, and blood pressure were also measured.
Type 2 diabetes was diagnosed based on results showing fasting plasma glucose ≥126 mg/dL, causal plasma glucose ≥200 mg/dL, or 2-h plasma glucose ≥200 mg/dl during a 75-g oral glucose tolerance test, or previous therapy for diabetes [53]. Hypertension was defined as systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, or taking treatment for hypertension. We defined dyslipidemia as the presence of LDL-C ≥140 mg/dL, HDL-C ≤40 mg/dL, TG level ≥150 mg/dL, or taking treatment for dyslipidemia.
Statistical analysis
The results were presented as median (interquartile range), unless otherwise stated. We used the Jonckheere-Terpstra test to compare the trend of data between three or more groups. The Cochran–Armitage test was used for the trend of the ratio between three or more groups.
Hepatic steatosis was graded as follows: with hepatic steatosis (L/S ratio <1.1) [11, 12], without hepatic steatosis (L/S ratio >1.296), and intermediate (L/S ratio = 1.1–1.296) [11]. In model 1, an ordinal logistic regression analysis was performed with L/S ratio as the objective variable and serum UA levels, plasma XOR activity, and HOMA-R as explanatory variables. In Model 2, BMI was added as an explanatory variable. In Model 3, we used an ordinal logistic regression analysis, and the L/S ratio was used as the objective variable; UA, XOR activity, and the HOMA-R were used as the explanatory variables, adjusted for age, gender, and components for Japanese diagnostic criteria of metabolic syndrome (AC, blood pressure, plasma glucose, HDL, and TG). Furthermore, HSI >36.0 were defined as high values [13], and logistic regression analyses was performed with HSI as the objective variables and UA, XOR, and HOMA-R as explanatory variables.
Statistical analyses were conducted using the BellCurve software version 2.15 (Social Survey Research Information Co., Ltd., Tokyo, Japan), with P <0.05 indicating statistical significance.