Hyperuricemia (HUA) is a group of heterogeneous chronic metabolic pathological conditions caused by purine metabolism disorder and/or uric acid excretion disorder. The excessive production and/or decreased excretion of uric acid level caused by any cause can lead to the occurrence of HUA. HUA is not only the direct cause of gout, but closely related to the onset and progression of many diseases such as diabetes, metabolic syndrome, chronic kidney disease, cerebrovascular-related diseases, and hyperlipidemia. Studies have confirmed that uric acid level is an independent risk factor leading to the progression of cardiovascular-related diseases such as hypertension and coronary heart disease, and it has an important impact on its prognosis (1). The latest research data show that the prevalence of HUA in the Chinese population has risen from less than 1.5% in the early 1980s to as high as 13% (2), and it is still rising and the incidence is showing a younger trend. Therefore, it is of great significance to study in depth the pathogenesis and further explore effective treatment measures of HUA.

Healthy people excrete uric acid in two ways mainly, of which 70% is excreted through the kidney, and the remaining 30% is excreted through the intestine (3). Intestinal microbes are a group of microorganisms designated to be planted in the intestinal tract, which have a symbiotic relationship with the human host and have a variety of important physiological functions such as participation in digestion, metabolism, promotion of nutrient absorption, synthesis of trace elements, and regulation of immune function. They are of great significance for the maintenance of our health (4). Researches in recent years have shown that changes in intestinal microbiota are not only related to diabetes, obesity, hypertension, and other diseases, but closely related to the occurrence of HUA. Studies have shown that changes in the gut microbiota and HUA promote each other and jointly aggravate the disease progression. For example, the intestinal microbiota can participate not only in the catabolism of purine and uric acid through the regulation of Escherichia coli and Lactobacillus (5–7), but also in the other two ways to affect the excretion of uric acid such as the production of short-chain fatty acids (SCFAs) (8–10) and changes in the number and distribution of uric acid transporters (11, 12) in the body. Besides, purine metabolism increases the production of uric acid, which can induce intestinal oxidative stress and then further promotes the infiltration of inflammatory cells and causes chronic inflammation in the intestinal tract. However, there are not many studies on the gut microbiota as an entry point to investigate HUA, and most of them are animal experiments. In view of the fact that most patients with HUA are asymptomatic and the intestinal microecology can change correspondingly in other disease states, it is more important and suitable to select the asymptomatic people who take physical examination as the research subjects to explore the correlation between HUA and gut microbiota. This study intends to explore the relationship among the serum uric acid (SUA) level, liver enzymes, and the detection of intestinal microbes in the physical examination population, and to provide more evidence support for finding the relationship among the serum uric acid, liver enzymes, and intestinal microecology.

It has been recognized that disturbances of the normal gut microbiome participate in the pathogenesis and progression of diverse chronic diseases, such as obesity (16), diabetes (17), hypertension (18), and liver cirrhosis (19), and the effect of gut microbiota in HUA has been explored gradually in recent years. For example, the current known interactions between the gut microbiota and HUA include that certain microbiota can regulate the synthesis and catabolism of purine and uric acid (such as Escherichia coli and Lactobacillus), produce some SCFAs or change the number and distribution of uric acid transporter. All of those studies have shown a strong correlation between the gut microbiota and HUA. However, most of them are animal experiments and the microbiota sequencing method is 16S rRNA, specific alterations of the gut microbiota composition and function between HUA (especially in males with HUA and high levels of liver enzymes) in asymptomatic people for physical examination have been rarely reported.

In view of the fact that most patients with HUA are asymptomatic and the intestinal microecology can change correspondingly in other disease states, our study is the first to select asymptomatic people for physical examination as the research subjects to explore the gut microbiome disturbances in males with HUA and high levels of liver enzymes and choose whole-genome shotgun sequencing technology, which are the two highlights and advantages of this research. Our results elucidated that there may do exist some targeted biomarkers of the gut microbiota and have the potential to be used as non-invasive, safe, and easy diagnostic tools for HUA. The new targeted biomarkers could be applied as a supplement to the traditional HUA diagnostic method to some extent.

In this study, we found that the level of SUA accompanied by WC, BMI, AST, ALT, GGT, Scr, and TG in the HUA group were significantly higher than that of the control group, while HDL was lower than the control group. Besides, the levels of FPG, blood pressure, TC, LDL, and inflammation indicators (WBC, PLT, NEUT, and NLR) were slightly, but not significantly, higher in the HUA group than in the healthy controls, TBIL and IBIL were of the opposite trend. Previous studies have confirmed that HUA is closely related to the occurrence of gout, metabolic syndrome, type 2 diabetes, hypertension, cardiovascular disease, chronic kidney disease, and so on, and it is an independent risk factor for the disease (20), which is consistent with our results. Numerous studies have shown that HUA is closely related to metabolic syndrome (obesity, hypertension, hyperlipidemia, insulin resistance), and the two factors are mutually causal and form a vicious circle (21–24). This comorbidity of chronic diseases suggests that there is a certain correlation between chronic diseases. Our results also suggest that HUA is associated with obesity and metabolism-related indicators, such as high levels of liver enzymes (5, 25), worse homeostasis in lipid and bilirubin metabolism, and poorer kidney function. Although the mean hepatic function was within the “current consensus normal reference range,” it had been already at a high level. Study has verified that when ALT exceeds 26 U/L, the occurrence of liver fibrosis increased, and it is suitable to adjust the reference range of liver function (26). In short, HUA is a metabolic disease related to habits, customs, age, gender, and inherited factors, and HUA is also correlated with obesity, hypertension, diabetes, renal failure, dyslipidemia, and liver diseases (27–29). Of course, the specific mechanism needs further study.

As for the gut microbiotas, we found that the gut bacterial diversity in HUA group was reduced significantly compared with that of healthy controls and the community of the microbiota was also of significant difference between the two groups. Just as Requena et al. (30) had mentioned the diversity of microbiota was closely associated with our health, the result of our study indicated that the gut microbiota may have altered significantly from a normal healthy status to the development of HUA. In the phyla level, compared with healthy controls, the abundance of Bacteroidetes in HUA group decreased [consistent with the previous research result (31)], while Firmicutes and Actinobacteria increased significantly. At the species level, totally 51 species were of significant difference after removing the low occurrence rate and low abundance species. Among them, 37 species were enriched in the healthy controls and 14 species were enriched in the HUA group. This suggests that maybe it is these significantly different populations in abundance that cause the significant changes in the composition of gut microbiota HUA.

Among the 51 species, compared with the species of healthy controls, the relative abundances of Bacteroidetes (Bacteroides uniformis, caccae, ovatus and plebeius, Alistipes finegoldii, senegalensis, and shahii); Firmicutes (Roseburia hominis, Ruminococcus callidus and lactaris, and Lachnospiraceae bacterium 3_1_46FAA); other (Adlercreutzia equolifaciens, Odoribacter splanchnicus, Butyricimonas synergistica, and Akkermansia muciniphila) decreased in the HUA group; while Firmicutes (Ruminococcus gnabus and torques, Lachnospiraceae bacterium 9_1_43FAA); Actinobacteria (Collinsella aerofaciens) increased. Interestingly, most of the gut microbiomes enriched in HUA group not only had significant positive correlations with the clinical characteristics, but also most of them are responsible for the production of SCFAs, while those microbiomes enriched in the healthy controls had highly negatively correlated with these clinical indexes.

In our study, some species that are known as SCFAs (e.g. butanoate) producer [Roseburia hominis (32, 33), Odoribacter splanchnicus (34), Ruminococcus callidus (35), Lachnospiraceae bacterium 3_1_46FAA (36), Bacteroides uniformis (37) and Butyricimonas synergistica (38)], together with the metabolic pathway of producing butanoate (CENTFERM-PWY and PWY-6590) were enriched in the healthy controls. Importantly, both of the two pathways had significantly positive correlations with the above bacterials. Especially, the bacterial of Odoribacter splanchnicus had the strongest correlation with them (r = 0.903 and 0.902 relatively) and had frequent correlations with clinical parameters, Roseburia hominis was the bacteria that most negatively correlated with clinical parameters and can produce large amounts of SCFAs, which play key protective roles against inflammation (39–41). As is known to all, Odoribacter splanchnicus is expressed in human intestine and its complete genome had been sequenced (42), and now it is recognized as a common butyric-acid producing bacterium, which has been reclassified from Bacteroides splanchnicus (34). In a recent study, Odoribacter splanchnicus together with Akkermansia muciniphila, has been associated with a healthy fasting serum lipid profile (43). The administration of prebiotics and the use of polyphenols have been stated as effective dietary strategies to modulate gut microbiota composition, which could induce the abundance of friendly bacteria, namely Akkermansia muciniphila, associated to health benefits in metabolic syndrome (44–46). Our study evidenced that HUA state may reduce or inhibit the growth of those “beneficial” bacterial members.

Besides, another relevant bacterium was Adlercreutzia equolifaciens, which was enriched in the healthy controls. A. equolifaciens is involved in metabolizing polyphenols and produced bioactive molecules involved in ameliorating metabolic disorders in obesity and diabetes (47). The study had verified that it can convert ingested isoflavones, which is abundant in legumes and soya beans, into equol (48). Equol has a high affinity for the estrogen receptor (49) and may be a selective estrogen receptor modulator. The incidence of HUA is high in men and postmenopausal women, and studies have concluded that it is the estrogen in premenopausal women that reduces its incidence through the follow ways, such as estrogen can directly affect the kidney excretion of uric acid by regulating transporters (49–51); inhibit the xanthine oxidase system (52); reduce the production of uric acid by maintaining the stability of lipid metabolism (53–55). Thus, we can propose Adlercreutzia equolifaciens may make contributions to alleviate the damage of HUA to our body in some estrogen-protection way. This finding is of great significance and maybe can provide a direction for the further exploration of new therapeutic targets for HUA based on gut microbiota.

On the contrary, Lachnospiraceae bacterium 1_4_56FAA, Ruminococcus gnavuswas and torques were enriched in the HUA group. Not only did they had positive correlations with most clinical indexes, but also the pathways that are significantly enriched in HUA group, such as superpathway biosynthesis of 5-aminoimidazole ribonucleotide (PWY-6122, PWY-6277 and PWY-6121), aromatic amino acids and chorismate synthesis (COMPLETE-ARO-PWY, ARO-PWY and PWY-6163), and L-lysine, L-threonine and L-methionine biosynthesis (PWY-724). Consistent with our results, all of the current studies have shown that the above three bacterials are closely related to immune inflammation in different diseases (56–60), though the inflammation indicators (WBC, PLT, NEUT, and NLR) were slightly, but not significantly, higher in the HUA group than in the healthy controls. Especially, R. gnavus can produce specific antigens and stimulates immune cells to produce corresponding antibodies, thus increasing inflammation (61, 62). As to the functional alterations, 5-aminoimidazole ribonucleotide is the key intermediate for purine nucleotide biosynthesis, the increased biosynthesis of 5-aminoimidazole ribonucleotide may increase the production of purines, thereby increase the level of uric acid. Besides, the aromatic amino acids and chorismate can act as substrates in other secondary metabolite pathways, such as indole derivatives and phenolic compounds, which are just the metabolites of our gut microbiotas. So far, no studies have been able to confirm the correlations between these structural and functional alterations of gut microbiota in HUA, but their significant alterations suggest that they may have important physiological significance in the occurrence and development of HUA, which is a research direction worthy for our further study.

There are some limitations in our study. First, this is a cross-sectional study, and it is not able to verify the causality between the discovered species and HUA, hence, further germ-free mice studies are still needed. Second, the study subjects come from the same region, thus, it is better to conduct a multicenter study from different regions owing to the influence of different regions and eating habits on the gut microbiota. Third, the gender in the HUA group is male adult, therefore, it is necessary to be cautious temporarily to apply the results obtained in this article to the female, minors or other population.

In conclusion, our study demonstrated the structural and functional alterations of gut microbiota in males with HUA and high levels of liver enzymes. In our study, the main clues of the correlation mechanisms between HUA and gut microbiome point to the pathways that can produce 5-aminoimidazole ribonucleotide, aromatic amino acids, and chorismate, which were enriched in the HUA group; the pathways that can produce SCFAs and the gut microbiotas that can produce SCFAs and equol were enriched in healthy controls. Generally, the potential mechanisms underlying the gut microbiome-Equol/SCFAs-uric acid accumulation link remains much for further exploration. The novel correlations between some microbiota species/pathways and uric acid accumulation could provide brand new directions for specific microbiota-targeted therapies.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ebi.ac.uk/ena,PRJEB48022.

The studies involving human participants were reviewed and approved by Ethics Committee from the First Affiliated Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study.

SD and SS: Conceptualization. JC, YW, and AL: Methodology and formal analysis. QQ, YZ, WL, TL, and XG: Resources. SS: Writing and original draft preparation. SD, LT, and AL: Writing and review and editing. JC and SY: Visualization. All authors have read and agreed to submit the manuscript.

This research was equally supported and funded by the Henan Province Medical Science and Technology Research Plan (LHGJ20200311 and LHGJ20200279), Chinese National Science and Technology Major Project (2018ZX10305410), Henan Province Youth Talent Promotion Project (Grant No.2021HYTP052), the Henan Province Postdoctoral Research Grant (001801005).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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