1. Introduction
Hyperuricemia is an abnormal condition characterized by increased serum concentrations of uric acid, and it is the cause of gout [
1]. It is defined as a condition in which the serum uric acid level exceeds 7.0 mg/dL, independently of sex and age [
2]. Gout is a progressive metabolic disease characterized by symptomatic hyperuricemia and deposition of monosodium urate crystals in joints and soft tissues. The prevalence of gout has increased globally [
3,
4,
5,
6], and its progression indicates an imbalance of uric acid synthesis and excretion [
7]. It has been suggested that hyperuricemia is in large part caused by decreased kidney excretion of uric acid [
8], reflecting the relative contribution of renal uric acid excretion to the maintenance of serum uric acid levels.
It is generally accepted that high protein diets increase the excretion of endogenous uric acid [
9]. In previous studies, the non-essential amino acid glycine increased urinary excretion of uric acid in rats and in several healthy human males [
10,
11]. Similarly, following oral administration of glycine in 12 gout patients for three days, decreased plasma uric acid levels were observed in eight cases and increased urinary uric acid excretion was observed in nine cases [
12]. These preliminary results suggest that continual ingestion of glycine effectively manages serum uric acid levels in hyperuricemic patients. Because little is known of the effects of other amino acids on uric acid excretion, we aimed to identify the amino acids that synergistically improve the effects of glycine on serum uric acid levels. In preliminary human studies, we observed transient declines in serum uric acid levels following oral administration of glycine with the essential amino acid tryptophan and found that the effect was optimal with comparatively smaller quantities of tryptophan (unpublished data). The present randomized, double-blind, placebo-controlled, crossover clinical trial was designed to confirm the serum uric acid-lowering effects of continual combined supplementation with glycine and tryptophan under conditions of mild hyperuricemia (serum uric acid levels of 6.6–7.9 mg/dL).
3. Results
From 178 recruited subjects, only 32 healthy subjects aged 21–62 years (47.9 ± 9.2) with mild hyperuricemia were randomly assigned to groups 1 or 2. One subject (No.28) was assigned to Group 2 and dropped out during period 1 in the crossover study due to concerns about chronic prostatitis. Therefore, final analyses were performed using data from 31 subjects (male, 30; female, 1). Rates of medication compliance, which was managed through the daily records of each subject, were 99.5% in period 1 and 99.7% in period 2. The characteristics of the subjects were shown in
Table 2.
Changes in serum uric acid concentrations were the main outcome variable during the trial, and these are summarized in
Table 3. Individual data of the subjects was shown in
Table S1 as Supplementary Material. In the present crossover trial, neither the carryover effect (
p = 0.911) nor the period effect (
p = 0.748) were significant for serum uric acid levels. Serum uric acid levels after 6-week treatments (6.7 mg/dL) were significantly lower than at baseline (7.1 mg/dL) in the active treatment group, and no changes in uric acid levels were observed in the placebo group (7.0 mg/dL before vs. 6.9 mg/dL after ingestion). These changes differed significantly between active (−0.4 mg/dL) and placebo-treated subjects (−0.1 mg/dL). Additionally, stratified analyses were conducted using the definition of hyperuricemia [
2], and similar results were obtained in both cases (> 7.0 and ≤ 7.0 mg/dL). Serum uric acid levels 6 weeks after ingestion were significantly lower than that before ingestion in the active treatment group, and no changes in serum uric acid levels were observed in the placebo group. Moreover, these changes indicated a significant difference between the active and the placebo-treated groups in both cases.
One hour urinary uric acid excretion, urate and creatinine clearances, urinary pH, and serum glycine and tryptophan concentrations are presented in
Table 4. No changes in urinary uric acid contents or urate and creatinine clearance were observed before and after ingestion in either active or placebo-treated subjects. After excluding one subject who exceeded the normal range at week 0, urate clearance in the active (
n = 30) group was significantly increased (
p = 0.027). No changes in urate clearance rates were observed in the placebo group. Similarly, urinary pH increased significantly after ingestion of amino acids, whereas no significant changes were observed in the placebo group. Serum glycine concentrations were increased after administration of amino acids and no such changes were seen in the placebo group. Both active and placebo groups had stable serum tryptophan levels before ingestion and after 6 weeks.
Multiple additional analyses were conducted to explore unknown physiological effects, and to confirm the safety of continuous ingestion of glycine and tryptophan mixtures for 6 weeks (
Table 5 and
Table 6). These analyses showed that platelet counts, serum creatinine and HDL-C levels changed significantly in the active treatment group. In addition, serum albumin and HDL-C levels changed significantly after ingestion of placebo. Serum triglyceride levels were also decreased significantly after ingestion of the active powder. In the present crossover trial, the carryover effect (
p = 0.021) was significant for serum triglyceride levels. Thus, detailed analyses were performed using the data during the Period 1 (0–6 weeks) as shown in
Table 6. Serum triglyceride levels 6 weeks later were significantly lower than before ingestion in Group 1 (active treatment group). The changes from baseline were significantly lower in Group 1 than in Group 2 (placebo group). Moreover, stratified analyses were performed using the diagnostic criteria of hypertriglyceridemia [
17]. We performed further analyses of serum triglyceride levels in subjects with levels of under 150 mg/dL and not less than 150 mg/dL before the trial. In these analyses, ingestion of the active mixture in Group 1 significantly decreased serum triglyceride levels compared with those in Group 2 in under 150 mg/dL. Statistical analysis was not performed for the subjects with hypertriglyceridemia (not less than 150 mg/dL) because the small sample size (
n = 3).
4. Discussion
Before planning this clinical trial, an animal study was conducted to identify amino acids that synergize with glycine to produce effective future functional foods for human consumption. In this study, combined doses of 2.0 g/kg glycine and 0.133 g/kg tryptophan significantly increased urinary urate excretion from Wistar rats after 7 h. In these experiments, cumulative uric acid excretion was 0.97, 1.07, 0.88, and 1.22 mg at 7 h after treatment with water, 2.0 g/kg glycine, 0.133 g/kg tryptophan, and 2.0 g/kg glycine with 0.133 g/kg tryptophan, respectively (unpublished data). Later, we confirmed that single combination doses of 3–6 g glycine with small quantities (0.1–0.4 g) of tryptophan transiently lowered serum uric acid more effectively than doses of glycine alone in a preliminary clinical study. The daily combined dosage was settled as an appropriate amount in continual supplementation.
Herein, we demonstrated that daily ingestion of 3.0 g of glycine with 0.2 g of tryptophan for 6 weeks decreases serum uric acid concentrations in subjects with mild hyperuricemia (serum uric acid, 6.6–7.9 mg/dL). Glycine is likely to increase urinary excretion of uric acid in rats and in healthy male humans [
10,
11]. Yet little is known about uric acid-lowering effects of tryptophan. It is clear from the present data that the mixture of glycine and tryptophan enhanced urate clearance in the cases, except for one wherein the subject had abnormally high urate clearance. Maximum serum levels of glycine or tryptophan would be achieved within an hour of ingestion, and would be followed by rapid decreases as the amino acids are metabolized. When we measured urinary uric acid levels, no changes followed ingestion of the active amino acid mixture, likely because our fasting measurements of urine parameters were taken after the changed serum and urine metabolite concentrations had returned to normal levels. The solubility of uric acid increases with an increase in pH [
18]. Alternatively, glycine has a buffering effect and is often used as an anti-acid agent. Potentially, increases pH due to daily ingestion of glycine and tryptophan for 6 weeks enhanced the solubility of urinary uric acid, and thus elevated urate clearance. Other mechanisms are also conceivable, and pyrazinamide, which is used to treat tuberculosis, reportedly disabled glycine-induced uricosuria [
19]. These effects of pyrazinamide may reflect enhanced urate reabsorption following exchange of its active metabolite [
20]. The urate transporter 1 (URAT1) is the primary re-absorptive urate transporter that is targeted by pyrazinamide [
21]. Thus, our results may reflect inhibition of the re-absorptive action of uric acid by URAT1. In this regard, it is inferred that tryptophan promote direct action of glycine against URAT1 because behavior of tryptophan to URAT1 is structurally difficult. Nonetheless, we suggest that, under the present conditions, increased uric acid excretion is primarily due to synergistic effects of glycine and tryptophan. Further studies are required to clarify the uric acid-lowering mechanisms of glycine and tryptophan.
In further experiments, the combined treatments with glycine and tryptophan decreased serum triglyceride levels, and this lowering effect was also observed in subjects with triglyceride level under 150 mg/dL. The supplementation of the amino acid mixture was likely to decrease serum triglyceride level in hypertriglyceridemia although the effect was uncertain due to few subjects. Both moderate and severe hypertriglyceridemia commonly reduces HDL levels and increases levels of atherogenic small dense LDL, and are associated with substantially increased long term total mortality and cardiovascular risk [
22]. To our knowledge, no previous reports show positive effects of glycine on serum triglyceride levels. In another study, however, supplementation with 15 g of glycine per day for three months did not affect serum triglyceride concentrations in patients with metabolic syndrome [
23]. In contrast, treatment with 1.0 g of L-tryptophan or 0.01 g of melatonin, a metabolite of tryptophan, for 6 weeks or 14 months reportedly decreased serum triglyceride levels and reduced concentrations of pro-inflammatory cytokines such as interleukin-1, -6, and tumor necrosis factor-alpha in patients with steatohepatitis or nonalcoholic fatty liver disease [
24,
25]. Hence, perhaps the triglyceride-lowering effects of mixtures of glycine and tryptophan are mediated by melatonin, which is a metabolite of tryptophan.
The safety of glycine and tryptophan treatments can be assumed because these amino acids are present in proteins and dietary supplements that are ingested in normal daily lives. Moreover, up to 90 g of glycine per day was administered over several weeks without serious adverse effects in clinical trials [
26]. Similarly, oral administration of up to 5.0 g of L-tryptophan per day did not cause any adverse effects in young adult females [
27]. During this clinical trial, no adverse effects were attributed to mixed glycine and tryptophan supplements. However, platelet counts and serum creatinine levels were significantly decreased by the present amino acid treatments, although these changes did not exceed normal values in any of our subjects. Collectively, this and other studies confirm the safety of oral treatments with the mixture of 3.0 g of glycine and 0.2 g of tryptophan.
With regard to the limitations of this study, the administration period of crossover regimens was only 6 weeks. Hence, longer trials may show greater benefits of these amino acids in subjects with mild hyperuricemia, and such studies would be best performed as parallel-group comparisons. Furthermore, male to female ratio indicated a disproportionate number. Further research might be needed, although our preliminary study had suggested that there was no effective difference between males and females by the amino acids supplementation.