Study design and participants.
This cross-sectional analysis was performed as part of the Hyogo Sleep Cardio-Autonomic Atherosclerosis (HSCAA) study47.48. The HSCAA study is a single-center cohort study that aims to investigate the interrelationships between sleep disorders, autonomic neuropathy, metabolic diseases and atherosclerotic diseases47.48. The HSCAA study included patients aged 20 years and older with one or more cardiovascular risk factors (obesity, smoking, history of cardiovascular events, hypertension, dyslipidemia, diabetes mellitus, chronic kidney disease) and treated at Hyogo College of MedicalHospital.
Since we started XOR measurements from 2018 for subjects who were enrolled or followed in the HSCAA study, this cross-sectional study included 310 patients, from January 2018 to July 2021, who consented to abdominal CT examinations. In the end, 223 patients were analyzed in the present study after excluding 87 with alcoholic habits (> 30 g/day for men and > 20 g/day for women), autoimmune hepatitis, viral hepatitis or under treatment with XOR inhibitors.
The HSCAA study was approved by the Hyogo College of Medicine Hospital Ethics Committee (Approval #2351). Written informed consent was obtained from all subjects and the study was conducted in full compliance with the Declaration of Helsinki. This study protocol has been approved by the Ethics Committee of Hyogo College of Medicine Hospital (Approval No. 3601) and performed with an opt-out option, as explained in the instructions posted on the website of the hospital. All methods in our study were performed in accordance with current guidelines and regulations.
Visceral fat area and subcutaneous fat area
Computed tomography was performed with SIEMENS SOMATOM Definition AS+ or SOMATOM Definition H (Siemens Healthcare GmbH, Erlangen, Germany) with 10 mm sections. We assessed visceral fat area (VFA), subcutaneous fat area (SFA), and waist circumference using Ziostation 2 (AMIN Ltd., Tokyo, Japan). AC was measured at the umbilical height.
Hepatic steatosis and hepatic fibrosis
L/S ratio and HSI were used to assess hepatic steatosis. Hepatic and splenic attenuation values were measured on non-contrast CT scans using four region of interest (ROI) circular cursors in the liver and two in the spleen. In the liver, four ROIs were located in each of the right and left lobes. All measurements were obtained manually in regions of uniform parenchymal attenuation, taking care to avoid vessels, artifacts, and other areas that might have falsely increased or decreased measurements. The calculation of the L/S ratio was as follows: L/S ratio = (Average liver attenuation value) / (Average spleen attenuation value)9,10,11,12. HSI was calculated from ALT, AST, BMI, gender and presence of diabetes mellitus13.
In addition, the NAFLD fibrosis score (NFS) and fibrosis index-4 (FIB-4) were calculated to predict the progression of liver fibrosis in patients with an L/S ratio 1,29,30,31,32. NFS was calculated from age, BMI, AST, ALT, presence of glucose intolerance, platelet count and albumin31.32. It has been reported that by applying the high threshold score (NFS > 0.676), the presence of advanced fibrosis can be diagnosed with high accuracy.31.32. The FIB-4 index was calculated from age, AST, ALT and platelet count. It has been reported that its threshold value 2.67 can predict liver fibrosis30.
XOR plasma activity measurement
The protocol for assaying XOR activity in humans has been reported previously26,27,28. Briefly, 100 μL of plasma samples (purified by Sephadex G25 resin) were mixed with a Tris buffer (pH 8.5) containing [13C2,15N2] xanthine as substrate, NAD+, and [13C3,15N3] AU as internal standard. The mixtures were incubated at 37°C for 90 min, mixed with 500 µL of methanol and centrifuged at 2000×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 by LC/TQMS. LC/TQMS included 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] AUs were also measured and production quantities were quantified from the calibration curve. XOR activities were expressed in pmol/mL/h26,27,28.
At the same time as the CT scan, blood samples were taken for AST, ALT, UA, fasting glucose, immunoreactive insulin, total cholesterol (T-Chol), high-density lipoprotein cholesterol (HDL-Chol), and TG . Additionally, serum UA levels were measured using the uricase/peroxidase technique with an automatic analyzer (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 blood glucose ≥ 126 mg/dL, causal blood glucose ≥ 200 mg/dL, or 2-hour blood glucose ≥ 200 mg/dL in a tolerance test oral glucose 75 g, or therapy for diabetes49. Hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or taking medication for hypertension. We defined dyslipidemia as the presence of LDL-C ≥ 140 mg/dL, HDL-C ≤ 40 mg/dL, TG levels ≥ 150 mg/dL, or taking treatment for dyslipidemia.
Results were presented as the median (interquartile range), unless otherwise specified. 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 ratio trend between three or more groups.
Hepatic steatosis was classified as follows: with hepatic steatosis (L/S ratio 11.12without 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 the 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 objective variable; AU, XOR activity and HOMA-R were used as explanatory variables, adjusted for age, gender and components of the Japanese diagnostic criteria for metabolic syndrome (AC, blood pressure, plasma glucose, HDL and TG ).
HSIs of >36.0, 13. Then, an ordinal logistic regression analysis was performed with HSI as objective variables and UA, XOR and HOMA-R as explanatory variables.
Statistical analyzes were performed using BellCurve version 2.15 software (Social Survey Research Information Co., Ltd., Tokyo, Japan), with P