Urate in Fingernail Represents the Deposition of Urate Burden in Gout
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www.nature.com/scientificreports OPEN Urate in fngernail represents the deposition of urate burden in gout patients Haibing Chen1,2,7*, Lili Zhao3,7, Fengjing Liu4, Si Chen4, Zhumeng Hu4, Lihui Chen4, Yiwen Ma4, Kaifeng Guo4, Aichang Ji5, Tony R. Merriman6 & Jun Zhe Min3* Urate in the fngernails of gout patients and healthy volunteers was successfully detected by high- performance liquid chromatography (HPLC) with ultraviolet (UV) in our previous research. This study aimed to further investigate whether nail urate could be a proxy for the burden of monosodium urate (MSU) crystals deposits in gout. To this end, we conducted a study in two parts. Firstly, we successfully detected urate in the nail by HPLC–UV and evaluated nail urate concentrations in control subjects and patients with gout. As expected, we found that levels of nail urate were signifcantly higher in patients with gout than in healthy controls, and the nail urate level was signifcantly correlated with the volume of MSU crystals deposits measured by dual-energy CT (DECT). Secondly, we found that nail urate can refect changes in urate levels in the body during urate lowering therapy through a 3-month follow-up study. Our results provide the possibility of quantifcation of urate in human fngernails as a non-invasive alternative for assessing MSU crystals deposits in gout. Urate is the end product of an exogenous pool of purines and endogenous purine metabolism1. Elevated serum urate levels (hyperuricemia) cause gout. Across the globe, both hyperuricemia and gout have increased in prevalence over the last few decades2,3. Many studies reveal that hyperuricemia is associated with many dis- eases, including diabetes mellitus 4, diabetic kidney disease 5, cardiovascular diseases6,7, stroke8, hypertension9, dyslipidemia10. Deposition of monosodium urate (MSU) crystals in the joint spaces and tissues is the fundamen- tal cause of gout. Long-lasting hyperuricaemia causes deposition of MSU crystals in the joints and sof tissues, triggering gouty arthritis and, if not properly treated, the formation of gouty tophi 11. Early diagnosis and treatment are essential for patients with gout. At present, the diagnosis of gout is mostly based on the classifcation criteria established by Te American College of rheumatology (ACR) in 1977 12. Although serum urate levels are recognized as the most important risk factor for gout, not all patients with hyperuricemia will develop gout13. In addition, during the acute attack of gout, serum uric acid levels may even be normal14–16. Moreover, the measurement of serum urate cannot assess the accumulation of urate deposits. In recent years, advanced imaging technology such as dual-energy CT (DECT) and ultrasound (US) have played an important role in the diagnosis of gout, which is refected in the 2015 classifcation criteria for gout formulated by Te American College of Rheumatology (ACR)17. Tese methods can reveal MSU crystals deposits to assess urate burden in gout. However, these techniques are ofen expensive and not readily available, thus leaving the clini- cian reliant on the patient history and presentation. Terefore, to accurately assess the seriousness of the disease, and initiate appropriate therapy, it is desirable to have a simple and reliable detection method of urate burden. In our previous research, we successfully detected urate in the fngernails of gout patients and healthy volun- teers by high-performance liquid chromatography (HPLC) with ultraviolet (UV)18. Here, we aimed to investi- gate the correlation between nail urate and the volume of urate deposits measured by DECT and to explore the 1Department of Endocrinology and Metabolism, Shanghai Eighth People’s Hospital, Shanghai 200233, China. 2Department of Endocrinology and Metabolism, Shanghai 10th People’s Hospital, Tongji University, Shanghai 200072, China. 3Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Pharmaceutical Analysis, College of Pharmacy, Yanbian University, Yanji 133002, Jilin Province, China. 4Department of Endocrinology and Metabolism, Shanghai Jiaotong University Afliated Sixth People’s Hospital, Shanghai 200233, China. 5Shandong Provincial key Laboratory of Metabolic Diseases, the Afliated Hospital of Qingdao University, Qingdao 266555, China. 6Department of Biochemistry, University of Otago, Dunedin, New Zealand. 7These authors contributed equally: Haibing Chen and Lili Zhao. *email: [email protected]; [email protected] SCIENTIFIC REPORTS | (2020) 10:15575 | https://doi.org/10.1038/s41598-020-72505-6 1 Vol.:(0123456789) www.nature.com/scientificreports/ possibilities of quantifcation of urate in human fngernails as a non-invasive alternative for assess MSU crystals deposits in gout. In addition, through a 3-month prospective observation study, we aimed to observe the efect of urate -lowering drugs on changes of nail urate levels. Methods Study population. Te male participants with gout (n = 82) and male healthy volunteers (n = 24) were recruited from the out-patient clinic at the Shanghai Clinical Centre for Diabetes (Shanghai, China). Te diag- nosis of gout was performed according to the 2015 Gout Classifcation Criteria 17. We excluded people who had fungal infections or other diseases that afect the normal state of the nail. Patients receiving regular urate- lowering therapy for more than 2 weeks were also excluded. Subjects with gout with febuxostat treatment (n = 25) or without febuxostat treatment (n = 9), and healthy volunteers (n = 4) were recruited from the out-patient clinic at the Shanghai Clinical Centre for Diabetes (Shang- hai, China) for a 3-month follow-up study. Patients with a history of anti-hyperuricemic drug use, acute kidney injury, malignant tumor, blood system diseases, application of glucocorticoid hormone and immunosuppres- sant therapy and hepatic impairment were excluded. Patients with gout were treated with febuxostat at a dose of 40 mg/day for 3 months. Blood urate levels were measured at 0 weeks, 2 weeks, 4 weeks, 8 weeks, 10 weeks, and 12 weeks, and nails were collected for nail urate detection. Te study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Com- mittee of Shanghai Jiao Tong University Afliated Sixth People’s Hospital. All study participants provided written informed consent prior to enrolment. Anthropometric and biochemical measurements. Demographic and clinical data, including age, sex, duration of gout, weight and height were recorded. BMI was calculated as weight in kilograms divided by squared height in meters (kg/m2). Te laboratory measurements used in this study have been described previously19. Venous blood samples were collected afer an overnight fast for measurements of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL- C), fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c), serum urea nitrogen, serum creatinine and serum urate on a Hitachi 7600 analyzer using an enzymatic assay (Hitachi, Inc., Tokyo, Japan). Determination of urate in fngernail. We used the methods reported in our previous study to collect human nail samples and to determine urate in human fngernail 18. In this study, the nails exposed to the edge of the fnger of the second, third and fourth fngers of the non-dominant arm were chosen for specimen collec- tion, and mixed them together for further processing. Nail samples were collected only once for each person. Briefy, the human fngernail samples were rinsed with 1.0 mL of 0.1% sodium dodecyl sulfate (SDS) for 1.0 min by ultrasonication. Te procedure was repeated another two times. Afer rinsing, SDS was removed by three washings with distilled water. Te fngernails were then dried in a desiccator under reduced pressure. Te dried fnger nails were crushed into a powder using a Shakeman 3 (BioMedical Science, Tokyo, Japan). A crushed fngernail sample (5.0 mg) was weighed into a 2.0 mL polypropylene tube. One hundred microliters of a 0.1 M NaOH solution was added. Te mixture was kept at 90 °C for 20 min to extract the urate, then vortex-mixed for 30 s and centrifuged at 3,000×g for 1.0 min, and repeated once. All the supernatant fuids was collected and 100 μL of an internal standard solution (100 μg/mL hypoxanthine) was added. Te resulting residues were added to 20 μL of 15% HCl for neutralization. Each 10 μL portion of the neutralization mixture was then subjected to HPLC–UV analysis. DECT examination. All subjects underwent DECT and the volume of MSU crystals was determined as previously described15. Briefy, DECT scans of the knees and feet were performed using two X-ray tubes and corresponding detectors (SomatomForce; Siemens Healthcare, Forchheim, Germany). In DE mode, these tubes scan at two diferent kilovolt levels, with simultaneous acquisition of two datasets, allowing for material-specifc diferences in the attenuation of the scanned tissue. Te scan parameters were as follows: detector collimation 2 × 128 × 0.6 mm and rotation time 500 ms. Te kilovolt level of tube A was set to 80 kV and the level of tube B was set to sn150 kV. Tube current parameters for feet were as follows: tube A,125mAs; tube B, 83 mAs. Trans- verse sections were reconstructed from the DE datasets, with kernel of Br40 and Qr40. Sagittal and coronal reformations in bone window settings were performed. Te transverse sof tissue kernel datasets of both tubes were loaded onto a SyngoVia Workplace (Siemens Healthcare, Forchheim, Germany) and reconstructed with a commercially available sofware program (CT DE Gout; Siemens Healthcare, Forchheim, Germany). Te total volume of urate crystal deposits was automatically quantifed in cubic centimetres by the sofware. If no tophus was visible, the DECT volume of the index tophus was defned as 0. Tis sofware uses automatic colour-coding visualization to detect MSU crystals. Te colour-coding datasets were reconstructed and reviewed in the trans- verse, sagittal and coronal image planes, with a slice thickness of 2 mm and an increment of 0.6 mm. All scans were obtained without intravenous contrast agent.