In this study, we utilized the causal associations between blood metabolites and gout through a bidirectional two-sample Mendelian Randomization (MR) analysis. We employed single nucleotide polymorphisms (SNPs) as instrumental variables (IVs) for this purpose. MR is a statistical methodology that utilizes genetic variants to establish causality, thereby enhancing our comprehension of the impact of specific genetic factors on disease susceptibility [12].We initially identified 31 candidate metabolites associated with gout risk through comprehensive genome-wide association study (GWAS) and MR analysis. After applying stringent Bonferroni correction, Benjamin-Hochberg method, and sensitivity analyses, we confirmed 1 metabolite with a Bonferroni-corrected P-value less than 1e-4 as significantly associated with gout risk, while 22 metabolites with Bonferroni-corrected P-values between 1e-4 and 0.05 were identified as potentially associated with gout risk, enhancing the robustness of our findings. The identification of these metabolites unveils potential pathological mechanisms and may provide new biomarkers and therapeutic targets for the prevention and treatment of gout. Notably, our data revealed that certain metabolites, such as X-11529, awere significantly associated with increased gout risk; conversely, metabolites like stachydrine, bradykinin, and piperine were associated with reduced gout risk. This might reflect the regulatory roles of these metabolites under pathological conditions on gout. By conducting reverse MR analysis on these associations, we further validated the causal directions of these relationships, strengthening the credibility of our study. These results underscore the potential roles of specific metabolites in the pathological process of gout. Overall, our study not only enhances the understanding of the association between gout and blood metabolites but also verifies the authenticity of these relationships through various statistical methods. As far as we are aware, there is limited research on this subject.Gout is an inflammatory arthropathy triggered by a disturbance in uric acid metabolism and is prevalent worldwide, with an increasing incidence in recent years. According to the Global Burden of Disease (GBD) Study, the prevalence of gout in the United States rose from 2007 to 2016 [25]. Gout is associated with socioeconomic status, being more common in lower-income populations. There are significant differences in prevalence and treatment patterns across different regions globally, with genetics, diet, and lifestyle all playing contributory roles. It is projected that by 2060, mortality related to gout may increase by 55%, posing a significant challenge to public health [26].The complex role of metabolites in the development of gout has garnered significant attention, particularly through the application of metabolomic technologies such as proteomics and lipidomics. These methods have been instrumental in identifying potential biomarkers linked to gout. Recent metabolic research has identified essential pathways and specific metabolites that regulate serum uric acid levels, a critical determinant in gout. By analyzing blood and urine metabolites, biomarkers for pathological states, particularly gout and hyperuricemia, can be detected [27, 28]. Notably, metabolites including uric acid, hypoxanthine, xanthine, guanosine, adenosine, Kynurenic Acid (KYNA), 2-Aminoethylphosphonic acid (2PY), DL-2-aminoheptanoic acid (2AMIA), and 5-hydroxyindoleacetic acid (5-HIAALB4) are proposed as biomarkers for gout [29,30,31]. Recent studies have shown significant changes in lipid, carbohydrate, amino acid, and energy metabolism in gout patients, highlighting the link between metabolic dysregulation and gout [32]. These findings open new avenues for diagnosis, prevention, and treatment, although the bidirectional causal relationships between specific metabolites and gout are yet to be established.Mannose, a carbohydrate closely related to glucose, plays a crucial role in protein glycosylation and energy provision. It is produced during the digestion of polysaccharides and glycoproteins, and notably influences the immune system by boosting antibody production against viruses [33]. Recent studies have established a significant link between mannose levels and gout. Metagenomic analyses have revealed notable differences in the gut microbiota composition between gout patients and healthy individuals, specifically highlighting an increased abundance of genes involved in mannose metabolism [34]. Additionally, the Chinese herbal medicine Wuwei Shexiang pill, recognized for its therapeutic potential in treating gout, may exert its effects by inhibiting several metabolic pathways including those of sugar and mannose, linoleic acid, and phenylalanine, thus mitigating symptoms of gout [35]. These findings underscore the critical role of mannose in the pathogenesis and management of gout, aligning with the risk effects observed in this study.Piperine, extracted from plants, offers bioenhancing and anti-inflammatory benefits. It shows promise in gout treatment by inhibiting the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, crucial for gout-related inflammation. This leads to reduced gout symptoms, prevents tophi, and protects cartilage. Piperine also decreases leukocyte infiltration, lipid peroxidation, and C-reactive protein production, aiding gout management [36]. Additionally, it increases cell membrane permeability, enhancing drug transport into cells [37]. Animal studies confirm that piperine alleviates urate-induced gouty arthritis in mice by reducing lysosomal enzymes, lipid peroxidation, tumor necrosis factor-alpha (TNF-α) levels, and boosting antioxidants [38]. Consistent with the analysis presented, piperine aligns as a protective factor against gout.Stachydrine from Motherwort exhibits notable anti-inflammatory effects, crucial for gout management [39]. It reduces key inflammatory cytokines like interleukin-1β (IL-1β) and TNF-α, impedes platelet-neutrophil aggregation, and inhibits neutrophil extracellular traps (NETs) formation, which are important in the pathology of gout [40]. It also curtails oxidative stress, aiding in osteoclast inhibition, anti-fibrosis, and tissue protection [41, 42]. Moreover, stachydrine provides osmoprotective advantages, reducing the risk of gout-related kidney damage [43]. Our findings, alongside existing studies, highlight stachydrine’s potential as a therapeutic for gout-induced bone and tissue damage, although further clinical trials are needed to validate its clinical benefits.1-Methylxanthine is a natural alkaloid akin to caffeine and theophylline,affects the central and cardiovascular systems. It is a caffeine metabolism byproduct [44]. Febuxostat inhibits xanthine oxidase (XO) to reduce uric acid levels, raising 1-methylxanthine’s excretion and effectiveness [45]. Studies confirm its importance in gout research; derivatives like 7-methylxanthine prevent urate crystal formation, potentially preventing gout attacks [46, 47]. A study found caffeine, as a prodrug of 1-Methylxanthine, links caffeine dose to 1-Methylxanthine/1-methyluric acid (1MU) ratio in urine, aiding gout treatment monitoring [48]. These insights offer new avenues for managing and treating gout.Our study, utilizing MR analysis, discovered that bradykinin may have a protective role in gout, diverging from previous observational studies that identified it as primarily pro-inflammatory [49, 50]. This discrepancy may be attributed to an insufficient control of confounding factors in earlier research [51]. Furthermore, we observed that the bradykinin receptor 2 (B2 receptor) decreases leukocyte infiltration and tissue damage, underscoring its critical role in gout therapy and its potential as a therapeutic target [52]. More research might elucidate the role and therapeutic value of bradykinin at various stages of gout, with the potential to offer novel strategies for gout management.A metabolomics study identified 23 serum metabolomic biomarkers differentially expressed across various stages of gout, predominantly associated with uric acid metabolism and oxidative stress [7]. The regulation of serum uric acid concentration is a complex process involving genetic, lifestyle, and environmental factors. Current data on the relationship between caffeine and gout or hyperuricemia are limited and controversial [53, 54]. Our results suggested that 1-methylxanthine might exacerbate the onset of gout through the caffeine pathway, consistent with prior literature. For instance, studies have indicated that excessive consumption of caffeine-containing coffee could elevate serum uric acid levels in healthy individuals, potentially due to caffeine influencing biochemical reactions (such as increasing the activity of xanthine oxidase) and modulating metabolic processes in the kidneys [55, 56]. A metabolomics analysis of Han Chinese individuals revealed that potential genetic targets related to gout are primarily enriched in the caffeine metabolism pathway, as well as the cysteine and methionine metabolism pathways [57]. However, the specific mechanisms and causal relationships require further investigation. This study identified two additional metabolic pathways closely related to gout: cysteine and methionine metabolism, and protein digestion and absorption. It highlights the critical role of amino acids in the production or excretion of uric acid, which is intimately connected to gout [58]. Furthermore, purines present in proteins can be metabolized into uric acid, affecting uric acid levels [59]. This research emphasizes the importance of further investigation into the metabolic pathways of caffeine and their relevance to biological functions associated with gout.The strengths of our research: Our research utilized MR to establish causality, relying on publicly available data, thus obviating the need for individual-level datasets. This methodological choice significantly reduces both time and financial costs associated with data acquisition. Our analysis diverges from traditional methods by employing a comprehensive, large-scale GWAS dataset to elucidate the relationships between metabolites and gout, offering insights that are unique in the field. Although our findings align with previous studies, our research boasts a more rigorous methodology. An earlier MR study [60], did not employ the Steiger test to exclude IVs indicating reverse causation, not did they conduct reverse MR analysis, potentially affecting the robustness of their findings. By contrast, we believe our study makes a valuable contribution and extension to the field through the use of a more stringent methodological approaches. Importantly, MR’s advantage over observational studies lies in its ability to minimize confounding variables, thereby enhancing the validity and reliability of our findings regarding the causal effects of metabolites on gout. To further validate these associations, we conducted detailed sensitivity analyses. These analyses specifically addressed potential issues such as horizontal pleiotropy and genetic confounding, ensuring the robustness and reliability of the causal inferences drawn from our study.However, there are limitations to our study. First, the GWAS data used are exclusively from European populations, which reduces the impact of population heterogeneity on the results but raises issues of generalizability. The conclusions may not necessarily apply to other populations. Further validation in more diverse populations using additional GWAS data is needed to confirm the generalizability of our results. Secondly, over half of the 31 candidate blood metabolites identified are unknown, indicating limited understanding and incomplete knowledge of gout pathogenesis. Further research is needed to elucidate their functions and biological roles. Thirdly, while our method is theoretically robust, further randomized controlled trials are crucial to verify causal relationships and translate findings into clinical applications, specifically in preventing or treating gout. Future research should address these limitations for a comprehensive understanding and reliable evidence.
