The metabolomics approach has been successfully applied in recent years to identify early signals or biomarkers of abnormalities[8], and to characterize biological pathways [9] and diagnose disease [8, 10]. LC‑MS is a powerful approach for the study of metabolomics due to its simple sample pretreatment process, wide coverage of metabolites, and high sensitivity [11]. In the present study, we used LC‑MS-based metabolomics to analyze metabolites in the serum of patients with AH and gout, to better understand predisposing factors for gout. We investigated alterations in the metabolome in AH and gout patients: significant alterations in FFAs, acylcarnitines, amino acids, bile acids, and lipids species were observed and could be useful to differentiate between HCs and HUA patients. Specifically, Glu, 2‑oxoglutarate, taurine, choline, and some non-essential amino acids might play important roles in the formation of urate. The process of urate formation also appeared to involve the metabolism of taurine and hypotaurine; Ala, aspartate and Glu; and D‑arginine and D‑ornithine. Although the metabolite changes were not significantly different between the AH and gout groups according to the PLS-DA score plot, the metabolic alterations observed might explain the occurrence of gout to some extent.
Certain amino acids participate in the biosynthesis of purine and subsequent formation of urates. For example, amino acids such as glutamine, glycine (Gly), and serine are implicated in the formation of urate in gout[12]. Therefore, it is reasonable to postulate that amino acids play important roles in the pathogenesis of gout. The serum levels of Ala, aspartic acid, oxoglutaric acid, glutamine, and L‑glutamic acid were higher in the HUA group. Ala, aspartate, and Glu are important in energy metabolism. In addition, glutamine and aspartate are important for the biosynthesis of endogenous purine nucleotide through the de novo synthesis pathway, which requires a significant amount of energy[13]. Glutamine and hypoxanthine are intermediary products in the formation of urate in birds, and it has been suggested that the mechanism may be the same in HUA patients. Therefore, glutamine may be an intermediate in the formation of both urea and urate[14]. Ts'ai-fan Yu reported an abnormally high plasma glutamic acid level in AH patients, and the elevated glutamate level was attributed to a deficiency of glutamic dehydrogenase[15]. In the presence of intracellular accumulation of Glu in cases of glutamic dehydrogenase deficiency, renal production of ammonium may be reduced due to its inhibitory effect on glutaminase. As a result of a renal blockade preventing ammonia formation, surplus glutamine may be instead be used for urate synthesis.
Oxoglutaric acid (2‑oxoglutarate), which can be produced from Glu by oxidative deamination (via glutamate dehydrogenase), is a key molecule in the tricarboxylic acid cycle and plays a fundamental role in determining the overall rate of this important metabolic process. Several other amino acids have also been shown to be differential metabolites, including arginine (Arg), leucine (Leu), isoleucine (Ile), and serine. Our findings are consistent with those of Kaplan et al.[16], who found significantly elevated levels of serum Ala, Ile, Leu, valine (Val), tyrosine (Tyr), Phe, and lysine (Lys) in patients with gout. Yű et al. [12] suggested that amino acids like glutamine, Gly, and serine are involved in the formation of urate in gout, as also observed in our study. An imbalance of amino acid homeostasis is closely associated with the formation of HUA.
Furthermore, many dipeptides were significantly changed in our HUA group, such as Arg-asparagine (Asn), Gly-Val, Leu-Gly, and Glu-Tyr. It has been reported that tryptophan-containing dipeptides inhibit xanthine oxidase (XO). Current understanding of dipeptides is incomplete, and other dipeptides may therefore be interesting targets for novel treatments and strategies for preventing HUA and related diseases.
Changes in taurine and hypotaurine metabolism were found in the HUA group. Compared to the HC group, the taurine level was significantly lower in the HUA group. Taurine is a sulfur amino acid, like methionine, cystine, cysteine, and homocysteine. It can be synthesized by the body from cysteine when vitamin B6 is present. Taurine efficiently decreased elevated XO activities and reduced urate formation in hyperuricemic rats. Moreover, it prevented any decrease in the mRNA and protein expression levels of urate transporters, and regulated renal urate excretion[17]. Therefore, taurine might be a promising agent for the treatment of HUA.
Previous studies found that HUA patients experienced changes in lipid metabolism similar to those seen during the course of cardiovascular disease[18]. The results of the present study suggested that abnormal fatty acid metabolism may be one of the metabolic pathways involved in the pathogenesis of HUA. Palmitic acid and oleic acid are involved in the synthesis and metabolism of fatty acids, elongation of fatty acids in mitochondria, synthesis of unsaturated fatty acids, and other biochemical reaction processes. The elevated plasma levels of these fatty acids in our patients with HUA suggested that disorders of fatty acid metabolism may occur in HUA. A marked alteration in HUA was related to the metabolism (β‑oxidation and transition) of FFAs. Compared to the HC group, most FFAs were significantly increased in the HUA group. FFAs are a key energy source, and β‑oxidation of fatty acids is reflected in the acylcarnitine profile[19].
Carnitine is essential for the transport of long-chain fatty acids from the cytosol to the intramitochondrial space in mammalian cells, and thus plays a major role in fatty acid oxidation [20]. Short-chain acyl derivatives of L‑carnitine also prevent the lipid peroxidation induced in various cardiovascular tissues by an excess of oxygen free radicals. In the presence of acetyl‑L‑carnitine, a significant reduction of XO activity has been detected[21]. According to the results of the present study, L‑carnitine levels in the HUA patients were reduced after the febuxostat treatment, while oleic, linolenic, and palmitate acids increased. Febuxostat is an XO inhibitor, and the increased oxidation of FFAs seems to be due to alteration of XO activity via effects on L‑carnitine.
Long-term elevation of urate leads to various complications, among which gout is the most common. In individuals with HUA, gout is caused by an inflammatory reaction that arises in response to the deposition of urate, in the form of MSU crystals, in articular joints and bursal tissues. In our study, compared to the gout group, the levels of Phe, aspartic acid, and kynurenine were significantly higher in the AH group.
Phe is an essential amino acid and the precursor of Tyr. Like Tyr, Phe is also a precursor for catecholamines including tyramine, dopamine, epinephrine, and norepinephrine. Phe is highly concentrated in a number of high-protein foods, including meat, cottage cheese, and wheat germ. An additional dietary source of Phe is artificial sweeteners containing aspartame. Aspartame, a low-calorie sweetener, was shown to have antipyretic, analgesic, and anti-inflammatory actions, and to delay osteoarthritis in animal models[22]. Disruption of rheumatoid factor activity by aspartame has been proposed to alleviate the pain and immobility associated with chronic inflammation of joints [23]. In 1981, a chemotactic factor having a molecular mass of 8,400 was identified in gouty synovial fluid. Amino acid analysis demonstrated that this chemotactic factor was relatively rich in aspartic acid, glycine, serine, glutamic acid, and Ala[24].
Kynurenine, a metabolite of tryptophan, was also significantly changed in our gout group. Tryptophan and its catabolites have been found to suppress T cell-driven local inflammatory responses through indoleamine 2,3‑dioxygenase[25]. Significantly elevated levels of one or more tryptophan metabolites were measured in the urine of active rheumatoid arthritis patients with comorbidities, especially hydroxykynurenine and kynurenine [26]. In summary, these results indicate that kynurenine may play an important role in the onset of gout-related inflammation. Moreover, aspartic acid may be involved in the chemotaxis of inflammation, and Phe is involved in the inflammation of joints.
The present study had some limitations. First, it included a small sample, and there was a bias about which patients had aspiration performed. Second, we could not investigate the relationship between plasma urate and serum metabolite levels due to a limited amount of data. Last, we did not examine the mechanisms underlying the metabolic alterations caused by HUA and gout.