Gout progression can be defined in gradual pathological stages: asymptomatic hyperuricemia without evidence of MSU crystal deposition, asymptomatic hyperuricemia with evidence of MSU deposition, MSU deposition with prior or current symptoms of acute gouty flares, and advanced gout characterized by tophi and chronic gouty arthritis 17. The pathophysiology of gout is based on the deposition of MSU crystals, which form in the presence of elevated SUA; i.e., hyperuricemia is the most critical predictor of gout. However, gouty flares do not develop in all individuals with hyperuricemia, and most cases of hyperuricemia remains asymptomatic 3,4. Epidemiological evidence indicates that there are modifying factors in the stages from normouricemia to asymptomatic hyperuricemia and from asymptomatic hyperuricemia to gout. In this study, a GC-MS/MS-based metabolomic investigation using both plasma and urine sample was conducted as a proof-of-concept study to identify differential features between gout and asymptomatic hyperuricemia.
Gout is associated with both environmental and genetic factors 18–23. GWAS and epidemiological studies identified several genes associated with gout 14–16,18,19, including the two most prominent urate transporters (ABCG2 and SLC2A9). Most GWAS compared gout to normouricemia (or without strict separation from asymptomatic hyperuricemia), and few GWAS investigated genetic factors associated with the progression from asymptomatic hyperuricemia to gout 24. Previous GWAS using participants with gout and asymptomatic hyperuricemia detected the loci of genes encoding urate transporters (ABCG2 and SLC2A9) as genetic factors aggravating normouricemia into gout 24. In addition, three genes (CNTN5, MIR302F, and ZNF724) were detected as ‘gout vs. asymptomatic hyperuricemia’-specific genetic factors 24. This suggests that mechanisms other than SUA elevation are involved in the progression from asymptomatic hyperuricemia to gout. In this study, we also investigated ABCG2 and SLC2A9 in participants with gout and asymptomatic hyperuricemia. Although the variant allele frequencies of the dysfunctional SNPs of ABCG2 (rs72552713 and rs2231142) were high, there were no differences in the frequencies of ABCG2 variants associated with SUA elevation between the gout and asymptomatic hyperuricemia groups (Table 3). The pathogenic variants of SLC2A9 were assessed in all participants, and the frequency of rs3733591, which has been reported to be related to SUA 25–27, was not significantly different between the gout and asymptomatic hyperuricemia groups. This result suggests that other factors including other urate transporters and environmental factors contribute to the progression from asymptomatic hyperuricemia to gout, although the urate transporters ABCG2 18,19,28−31 and SLC2A9 32,33 play an important role in regulating SUA.
Additionally, a previous network analysis identified a cluster of genes involved in glucose metabolism as gout-related factors 14. Our metabolomic analysis also suggested that carbohydrate metabolism is enhanced in high-risk gout (Fig. 2a, b). This result was consistent with that of the previous study. Several studies found that plasma glucose is positively and negatively related to SUA and urinary urate clearance, respectively, via insulin resistance. 34–37. Insulin is the hormone that promotes the utilization of glucose and lowers blood glucose levels. Although insulin concentrations were not measured in this study, the levels of glucose, TCA cycle intermediates, and pyruvate and lactate, which are the main products of glycolysis, tended to be higher in patients with gout. In particular, lactate is interpreted as a marker of typical anaerobic glycolysis, and its accumulation usually implies a high energy demand in biological systems; e.g., glycolysis activation accompanied by hypoxia or inflammation increases the accumulation of lactate 38. In the present study, plasma lactate levels tended to be higher in the gout group, suggesting that the accumulation of lactate reflects the low utilization and/or increased intake of glucose and facilitates the induction of gouty flare via decreased pH. In the gout group, 2-ketoglutarate, isocitrate, and malate tended to be accumulated compared to the findings in the asymptomatic hyperuricemia group (Tables 4 and 5). The accumulation of these TCA cycle intermediates requires the reduction of nicotinamide adenine dinucleotide (NAD+) for subsequent reductions. By contrast, the plasma levels of other TCA cycle intermediates (citrate, succinate, and fumarate) that do not require NAD + reduction were not significantly different between the gout and asymptomatic hyperuricemia groups (Tables S1 and S2). This imbalance also suggests that gout onset is at least partially related to carbohydrate metabolism, including mitochondrial internal respiration. Acute gouty flare is initiated by synovial resident macrophages, which are innate immune cells. The deposition of MSU crystals induces nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome formation and mediates localized acute inflammation 39. Although the association between mitochondria function and NLRP3-associated inflammation such as gout has been reported 40,41, it remains controversial because NAD+, which reflects mitochondrial internal respirational function, was not measured in this study.
It has been reported that the main pathology of hyperuricemia in patients with gout is decreased urate excretion in the kidneys and intestine 28,42. The regulation of urate excretion, especially by a cluster of urate transporters in the kidneys, is important in the homeostasis of SUA 43, which has been corroborated by the presence of type I and type II renal hypouricemia caused by SLC22A12 44–46 and SLC2A9 dysfunction 32,47, respectively. In urate reabsorption, SLC22A12, which is expressed at the apical membrane of proximal tubule cells, transports urate to inside the cell in conjunction with the outward transport of monocarboxylate (Fig. 2d). In addition, SLC5A8 transports monocarboxylate, which is essential as the counterpart in urate reabsorption, from urine into proximal tubule cells 48. Namely, SLC22A12 and SLC5A8 act cooperatively in urate reabsorption mediated by monocarboxylate. Indeed, it has been reported that excess blood lactate is excreted in exchange for reabsorbed urate 49. Of note, urinary nicotinate was identified as the metabolite that most prominently differentiated gout from asymptomatic hyperuricemia in this study (Tables 4 and 5). Although we cannot discuss causality because this was a non-interventional and cross-sectional observation, this result might reflect the association between gout and the accumulation of monocarboxylates including lactate and nicotinate, the renal excretion of which is coupled to urate reabsorption.
Because this was a pilot study, several problems and issues must be addressed for future full-scale metabolomics studies. First, the sample size must be increased to distinguish low-risk gout from asymptomatic hyperuricemia. The reason is that the pathophysiology of gout is based on asymptomatic hyperuricemia, and the metabolic features of these conditions are similar. The second is consideration of the duration of hyperuricemia. Naturally, the cumulative risks of progression to gout increases with the duration of hyperuricemia 3. Therefore, a more sensitive discriminant analysis might be feasible by comparing individuals who remain asymptomatic despite a long duration of hyperuricemia and patients with gout who experience flares despite a short duration of hyperuricemia. Because this study did not include many such individuals, this is proposed as a future issue. Third, genetic factors other than urate transporters should be investigated. Although we found that carbohydrate metabolism was perturbed in patients with high-risk gout, we did not clarify whether this is attributable to genetic or environmental factors. Our pilot study identified glucose metabolism factors that have been implicated in gout as genetic factors to be investigated in future full-scale studies 14. Finally, we must acknowledge the limitations of our results regarding confounders and generalization. Six patients with gout received uric acid-lowering medications within 2 weeks of enrollment. It is undeniable that the presence or absence of this external intervention might have affected the results of this study. In addition, the entire discovery dataset in this study consisted of only male participants, which limits the generalizability of the findings. Despite these limitations, this study comprehensively profiled both plasma and urinary metabolism and attempted to differentiate gout and asymptomatic hyperuricemia. In conclusion, our pilot study suggests that glycolysis compounds, TCA cycle intermediates, and urinary nicotinate, which are related to urinary urate excretion, are potential biomarkers distinguishing gout from asymptomatic hyperuricemia.