Urate in fingernail represents the deposition of urate burden in gout patients

Urate in the fingernails 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 significantly higher in patients with gout than in healthy controls, and the nail urate level was significantly correlated with the volume of MSU crystals deposits measured by dual-energy CT (DECT). Secondly, we found that nail urate can reflect changes in urate levels in the body during urate lowering therapy through a 3-month follow-up study. Our results provide the possibility of quantification of urate in human fingernails as a non-invasive alternative for assessing MSU crystals deposits in gout.


Determination of urate in fingernail.
We used the methods reported in our previous study to collect human nail samples and to determine urate in human fingernail 18 . In this study, the nails exposed to the edge of the finger of the second, third and fourth fingers of the non-dominant arm were chosen for specimen collection, and mixed them together for further processing. Nail samples were collected only once for each person. Briefly, the human fingernail samples were rinsed with 1.0 mL of 0.1% sodium dodecyl sulfate (SDS) for 1.0 min by ultrasonication. The procedure was repeated another two times. After rinsing, SDS was removed by three washings with distilled water. The fingernails were then dried in a desiccator under reduced pressure. The dried finger nails were crushed into a powder using a Shakeman 3 (BioMedical Science, Tokyo, Japan). A crushed fingernail 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. The 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 fluids was collected and 100 μL of an internal standard solution (100 μg/mL hypoxanthine) was added. The 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 described 15 . Briefly, 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 different kilovolt levels, with simultaneous acquisition of two datasets, allowing for material-specific differences in the attenuation of the scanned tissue. The scan parameters were as follows: detector collimation 2 × 128 × 0.6 mm and rotation time 500 ms. The 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. Transverse sections were reconstructed from the DE datasets, with kernel of Br40 and Qr40. Sagittal and coronal reformations in bone window settings were performed. The transverse soft tissue kernel datasets of both tubes were loaded onto a SyngoVia Workplace (Siemens Healthcare, Forchheim, Germany) and reconstructed with a commercially available software program (CT DE Gout; Siemens Healthcare, Forchheim, Germany). The total volume of urate crystal deposits was automatically quantified in cubic centimetres by the software. If no tophus was visible, the DECT volume of the index tophus was defined as 0. This software uses automatic colour-coding visualization to detect MSU crystals. The colour-coding datasets were reconstructed and reviewed in the transverse, 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.
Statistical analysis. All statistical analyses were carried out using SPSS version 17.0 (SPSS, Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation, and compared by two-sided t-tests or, if the data were not distributed normally, variables were expressed as median with interquartile range and the Mann-Whitney U-test was used. Categorical variables were expressed as percentages and using a chi-squared test or Fisher's exact test. Correlations among the study variables were tested by the Pearson's correlation coefficient. Those parameters with P-value < 0.05 in the Pearson's correlation analysis were subsequently entered www.nature.com/scientificreports/ into multiple linear regression analysis with nail urate as a dependent variable. A P-value < 0.05 (two-sided) was considered statistically significant.

Results
Clinical characteristics of control subjects and patients with gout. Baseline characteristics of 106 subjects are shown in Table 1. There was a statistically significant difference in age between the groups (P < 0.05). However, after controlling for age, patients with gout had significantly higher body mass index (BMI), fasting glucose (FPG), blood pressure, trigylcerides (TG), serum creatinine levels and a higher frequency of diabetes, hypertension and non-alcoholic fatty liver disease (NAFLD) than those without gout. Meanwhile, urea nitrogen (BUN), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) did not differ between the groups.

Comparison of fingernails and serum urate between control participants and gout.
We examined urate concentrations in fingernails and serum of all 106 study participants. Figure 1 shows the HPLC-UV chromatograms obtained from urate in the fingernails from the control subjects and gout. The peaks corresponding to the urate were completely separated without any interference from the endogenous substances in the human fingernails. Using this method, the amounts of urate in the fingernail of the control subjects and gout were determined. As expected, urate concentration in the serum was significantly higher in the gout group compared to the control group (P < 0.001, Table 1). Moreover, we found that levels of nail urate was also significantly higher in patients with gout (median 170.4 ng/mg; interquartile range: 120.0-570.3 ng/mg fingernails) than in healthy controls (median 50.0 ng/mg; interquartile range 32.9-82.9 ng/mg, P < 0.001, Fig. 2A). Next, we investigated the relationship between serum urate and nail urate. The analysis demonstrated a significant positive association between serum urate and nail urate (r = 0.27; P = 0.01, Fig. 2D). Although this correlation is weak, these results suggest that a significant linear relationship may exists between urate concentrations in serum and fingernails. They also suggest the possibility of estimating urate concentrations in serum from that in fingernails, and potentially replacing blood collection with non-invasive fingernail collection.
Relationship between nail urate levels and volume of MSU crystals. We investigated the correlation between nail urate and the volume of MSU crystals measured by DECT by testing the relationship of nail urate with a cluster of anthropometric parameters and biochemical indices include the volume of MSU crystals measured by DECT (Table 2). Pearson's correlation analysis showed that the nail urate was positively correlated with serum urate (r = 0.27, P = 0.01), the volume of MSU crystals (r = 0.42, P < 0.001) (Fig. 2B), duration of gout (r = 0.35, P = 0.011), SBP (r = 0.30, P = 0.005), DBP (r = 0.26, P = 0.014) and fasting plasma glucose (r = 0.27, P = 0.041), but not correlated with age (r = − 0.03, P = 0.793) in all 106 subjects. The multiple linear stepwise Table 1. General and clinical parameters of control subjects and Gout. Data are expressed as means ± SD. Data were analyzed using independent-sample t test and covariance analysis (adjusted for age). SBP systolic blood pressure, DBP diastolic blood pressure, BMI Body Mass Index, FPG fasting plasma glucose, TC total cholesterol, TG triglycerides, HDL-c high density lipoprotein cholesterol, LDL-c low-density lipoprotein cholesterol, BUN Urea nitrogen, Cr Creatinine, NAFLD non-alcoholic fatty liver disease. # P < 0.05 vs. control; *P < 0.05,**P < 0.01, ***P < 0.001 vs. control; adjusted for age.

Effect of febuxostat therapy on nail urate levels in people with and without gout. Supplemen-
tary Table S1 summarizes the anthropometric characteristics and metabolic variables at baseline. After 3 months of follow-up, patients with gout who received febuxostat had a significant decrease in serum urate levels compared with healthy controls and untreated group (P < 0.001, Fig. 3A). Moreover, we found that the nail urate level in the treatment group also decreased significantly (P < 0.05, Fig. 3B). As the treatment progressed, the nail urate gradually declines (Fig. 4C), but there is no obvious change in the healthy controls group (Fig. 4A) and the untreated group (Fig. 4B). These suggest that nail urate can also reflect changes in urate levels in the body during urate-lowering therapy.

Discussion
The human fingernail may serve as a non-invasive alternative to estimate MSU crystals deposits in gout. In the present study, 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 significantly higher in patients with gout than in healthy controls and we found a strong positive association between nail and serum urate. Moreover, we found that nail urate can reflect changes in urate levels in the body during urate lowering therapy. Importantly, the novelty of our study is that we demonstrated for the first time that the nail urate level was significantly correlated with the volume of MSU urate deposits measured by DECT. Our results www.nature.com/scientificreports/ provide the possibility of quantification of urate in human fingernails as a non-invasive alternative for assessing MSU crystals deposits in gout. Although identification of MSU crystals within a tophus sample or a joint aspirate on light microscopy is the gold standard for the diagnosis of gout, the diagnostic confirmation with imaging is becoming more attractive for clinicians and patients due to its noninvasiveness 17 . Both DECT and ultrasound can detect MSU crystal deposition, and have shown good diagnostic accuracy, especially useful in patients with longer duration of gout. However, these technologies still have certain shortcomings in terms of efficiency and cost, and there is still a need to explore simpler and more convenient detection methods to diagnose and assess the severity of gout 15,20 . Human nails, as a non-invasive biological sample, have recently attracted attention. Compared with other biological samples, nail samples make up for the limitations of blood, urine, saliva and other samples due to its slow growth and can reflect the longer-term exposure of disease or post-treatment status. Recently, studies have reported that physiological information can be obtained through nails and may be used as a non-invasive diagnostic biological sample for chronic diseases [21][22][23][24] . Tirado-González et al. performed a nail biopsy on a person who identified the presence of a fungal infection and serendipitously discovered the presence of MSU urate crystals in the nail of a gout patient 25 . Li et al. used HPLC-UV to detect the amount of urate in the human fingernail in 26 healthy controls and 22 gout patients, and the nail urate concentration was found to be significant different between the two populations. The above results suggest that the presence of urate can be detected in the nail and there is a significant difference between gout and healthy controls. The content of urate in the nail may represent long-term average urate level and a proxy for level of MSU crystals deposited in the tissue during a certain period of time, which may be several months because fingernails grow about 3 mm/month or 0.1 mm/day; toenails about 1 mm/month, with complete replacement achieved in 6-9 months [26][27][28] . At present, the mechanism of urate www.nature.com/scientificreports/   www.nature.com/scientificreports/ deposition in nails is still unclear, and it has not been reported how long the nail urate level can reflect the serum urate level. Further research is needed in the future.
In this study, we found that the nail urate level was significantly correlated with volume of urate deposits measured by DECT.This result indicated higher nail urate was a predictive factor of the volume of MSU crystals. While hyperuricemia is a strong risk factor for gout, our study did not find a significant association between serum urate level and MSU crystals deposits measured by DECT, which indicated that measurement of serum urate does not assess the accumulation of urate deposits and therefore cannot estimate the urate burden. The result that the duration of gout was independently correlated with the volume of urate deposits measured by DECT further conformed the previous findings that the duration of gout and serum urate level were significantly associated with the urate crystal volume.
This study has some limitations. First, the small sample size, and there was also a bias for patients in whom aspiration was performed. Second, the analyses were limited to the knees and feet. This was because there has not been a general consensus so far on which joints should be examined to assess urate burden in gout and the knees and feet are known to have more prevalent MSU crystals deposits identified by DECT. Third, we measured the serum urate value only once, due to the fluctuation of serum urate, the average value of multiple measurements may be a better assessment of the relationship between serum urate and nail urate and the volume of MSU. Finally, compared with serum urate detection, the cost of HPLC-UV detection is relatively expensive, and in the future, better optimization is needed in order to be less expensive, convenient to operate, and suitable for routine analysis.

Conclusions
In conclusion, we found in the present study that the nail urate concentration was positively correlated with the volume of MSU crystals as measured by DECT in males with gout and that nail urate can also reflect changes in urate levels in the body during urate-lowering therapy. At present, there is no good quantitative index of wholebody MSU crystal deposition. Although serum urate is an important indicator for the immediate change of urate level in the body, nail urate can reflect changes in urate deposition in the body during urate-lowering therapy and determination of nail urate may provide useful information for management of gout in situations where blood sample analysis and image technology are not possible. In future, a large, prospective, multicentre trial is needed to assess the performance of this biomarker and to demonstrate its effectiveness in clinical practice when compared to currently accepted methodologies.