Acute effect of green tea catechins on uric acid metabolism after alcohol ingestion in Japanese men

Alcohol consumption is associated with hyperuricemia and gout. Previous studies have indicated a role for green tea catechins in uric acid (UA) metabolism. This study aimed to elucidate the acute effect of green tea catechins in terms of enhancing urinary excretion of UA and xanthine/hypoxanthine (Xa/HX; UA precursors) after alcohol ingestion. In a randomized crossover study, ten healthy Japanese subjects consumed test meals, including a Japanese distilled spirit (Shōchū) with water (SW) or Shōchū with catechin-rich green tea (SC), each containing 20 g of alcohol. The SC contained 617 mg of catechin in total. Serum and urine UA and Xa/HX concentrations were measured. Blood samples were collected after 2.5 h, and urine samples were collected between 0 and 5 h after consuming the test meal. Urine UA and Xa/HX excretions were significantly higher in the SC group than in the SW group (UA: SW, 0.45 ± 0.08; SC, 0.52 ± 0.09; Xa/HX: SW, 0.08 ± 0.04; SC, 0.16 ± 0.05 mg/kg/h). UA clearance (CUA) and fractional UA excretion (FEUA) tended to increase more in the SC group than in the SW group (CUA: SW, 7.76 ± 2.14; SC, 8.75 ± 2.23 mL/min/1.73 m2; FEUA: SW, 6.08 ± 1.36; SC, 6.64 ± 1.42%). No significant differences in serum UA and Xa/HX concentrations were observed between two groups. It was concluded that green tea catechins can enhance the excretion of UA and Xa/HX, even though alcohol is ingested. UMIN000040076. Retrospectively registered 7 April 2020. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000045687 Key Points • Green tea catechins enhance the excretion of uric acid and xanthine/hypoxanthine, even when alcohol is ingested simultaneously. • In case of non-adherence of limiting alcohol intake, catechin-rich green tea may be an effective dietary component to continue dietary therapy. Key Points • Green tea catechins enhance the excretion of uric acid and xanthine/hypoxanthine, even when alcohol is ingested simultaneously. • In case of non-adherence of limiting alcohol intake, catechin-rich green tea may be an effective dietary component to continue dietary therapy.


Introduction
Hyperuricemia, which is the main risk factor for gout, is defined as serum uric acid (UA) concentration of ≥7 mg/dL. The prevalence rates of hyperuricemia and gout continue to increase [1]. Moreover, hyperuricemia is also closely associated with metabolic syndrome, chronic kidney disease, and cardiovascular disease [2][3][4][5][6]. Several studies have reported that lifestyle factors, including alcohol, purine-rich foods, and fructose, are associated with the development of hyperuricemia and gout [7,8]. Thus, lifestyle improvement may be important for preventing hyperuricemia.
Alcohol consumption has been associated with the development of gout [9]. Adenosine triphosphate is consumed during alcohol metabolism, followed by adenine nucleotide degradation. As a result, the plasma concentrations of purine bases, such as hypoxanthine (HX), xanthine (Xa), and UA, increase [10,11]. Furthermore, alcohol consumption increases the lactate concentration in the blood, interfering with urinary excretion of UA [10]. Therefore, alcohol consumption results in increased serum UA concentrations. It was reported that allopurinol, a clinically used xanthine oxidase (XO) inhibitor prescribed for gout, seems to be effectively controlling the rapid increase in plasma UA concentrations following beer ingestion [11]. However, few studies have evaluated the potential of food components on UA metabolism after alcohol consumption.
Tea is widely consumed in the world. Although approximately 78% of the tea production worldwide is black tea [12], green tea is widely consumed, especially in Asian countries; we primarily focused on green tea in this study. Green tea contains polyphenols, particularly catechins. The major catechins present in green tea are catechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. It has been reported that green tea polyphenols decrease serum UA concentrations by regulating XO activity and urinary excretion of UA in hyperuricemic mice [13][14][15]. In addition, green tea extract lowers serum UA concentrations in healthy individuals to some extent [16]. These studies together indicate a role for green tea catechins in UA metabolism. However, these studies evaluated the chronic effect (≥7-day ingestion) of green tea catechins, and the acute effect of green tea catechins in UA metabolism remains unclear. Furthermore, a recent meta-analysis showed that green tea consumption was positively associated with serum UA concentrations due to the limited number of studies (three studies) [17]. Thus, the effects of green tea remain unclear, and the acute effect of green tea catechins for enhancing the urinary excretion of UA after alcohol ingestion remains to be understood. Since humans do not have a uricase, which is only found as a non-functional pseudogene in the human genome [18], it is necessary to elucidate the physiological response in human intervention studies.
Therefore, this study aimed to elucidate the acute effect of green tea catechins on UA metabolism after alcohol ingestion.

Subjects
Ten healthy Japanese men were recruited in this study. Exclusion criteria were liver or renal dysfunction, alcohol dependence, and receiving treatment for hyperuricemia or gout. Subjects who had alcohol intolerance were also excluded. The clinical and biological characteristics of the subjects are shown in Table 1. The mean values ± standard deviation (SD) of age and body mass index (BMI) were 25.0 ± 4.5 years and 22.1 ± 2.4 kg/m 2 , respectively.
The present study was performed after obtaining written informed consent from all subjects and was approved by the Ethics Committee of the University of Shizuoka and regist e r e d w i t h U M I N ( U M I N r e g i s t r a t i o n n u m b e r : UMIN000040076). The study was performed in accordance with the Helsinki Declaration.

Study protocol
We used a randomized crossover study design. The experiment was conducted so that the test days were separated by a washout period of at least 7 days. All the subjects were asked to avoid heavy exercise and any intake of alcohol and purinerich foods (>200 mg/100 g) for 3 days prior to each study day. All the subjects were instructed to eat from 20:00 to 21:00 h prior to each test day. After an overnight fast, the subjects were provided with the same prescribed foods (breakfast) at 08:00 h and were required to consume breakfast within 20 min. At first visit, the body weight and height were measured. The subjects were instructed to abstain from foods and beverages other than those prescribed from 08:00 to 13:00 h. The subjects were provided with their test meals (lunch) at 13:00 h and required to consume each test meal within 20 min. During the experimental period, all subjects were instructed to drink water, 100 mL/h. All subjects underwent a 5-h urine collection from 13:00 to 18:00 h. Venous blood samples were collected

Test meals
Two different test meals were used: a Japanese distilled spirit (Shōchū) with water (SW) and Shōchū with catechin-rich green tea (SC). The SW and the SC each contained 20 g alcohol and were made up to produce a final total volume of 500 mL. We used catechin-rich green tea, which is a commercial beverage, and the SC contained 617 mg of total catechin. All test meals were ingested with 60 g steamed chicken and 10 g sesame dressing ( Table 2). Steamed chicken contained about 85 mg/60 g purines and Shōchū did not contain purine according to the guidelines for the management of hyperuricemia and gout in Japan [19].

Blood and urine analysis methods and anthropometric measurements
Blood samples were centrifuged at 2400 rpm for 10 min at 4°C and separated into serum and stored at −80°C until analysis of serum creatinine (Cre), UA, Xa/HX, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transpeptidase, and blood urea nitrogen concentrations. Urine samples were used for analysis of pH, Cre, UA, and Xa/HX concentrations. The analyses of serum and urine samples were performed by a blood test company, SRL, Inc. (Tokyo, Japan), except for the analyses of urine pH and Xa/ HX concentrations. The pH was measured using a portable pH meter (LAQUA act, D-71, Horiba Scientific, Kyoto, Japan). The concentration of Xa/HX was measured using a Xa/HX colorimetric assay kit (Bio Vision, USA). Anthropometric measurements were determined using a bioelectrical impedance analysis method (innerscan DUAL RD-909, TANITA Corporation, Tokyo, Japan). Height was measured using a stadiometer.

Calculating formulas
We calculated the creatinine clearance (Ccr), filtered UA load (F UA ), UA clearance (C UA ), urinary UA excretion per kilogram of body weight per hour (U-UA excretion), and renal fractional UA excretion (FE UA ) using the following formulas (U denotes urine, S denotes serum, BSA denotes body surface area, and BW denotes body weight) [19,20]: We also calculated filtered Xa/HX load (F Xa/HX ), Xa/ HX clearance (C Xa/HX ), urinary Xa/HX excretion per kilogram of body weight per hour (U-Xa/HX excretion), and renal fractional Xa/HX excretion (FE Xa/HX ) using the following formulas:

Statistical analysis
All data are shown as means ± SD. The Shapiro-Wilk statistic was used for data normality testing. Parametric analysis was used for normal distribution data, and non-parametric analysis was used for data exhibiting a non-normal distribution. Differences between the SW and SC groups were identified using a paired t test or the Wilcoxon signed-rank test. Probability (P) values less than 0.05 were considered statistically significant in all analyses. Statistical analyses were performed using SPSS for Windows, release 26.0 (SPSS, Chicago, IL).

Serum UA and Xa/HX concentrations
The S-UA concentration in the SW group was 6.3 ± 1.0 mg/ dL, and in the SC group, it was 6.3 ± 0.9 mg/dL. The S-Xa/ HX concentration in the SW group was 11.7 ± 12.7 μg/mL, whereas 11.4 ± 12.2 μg/mL in the SC group. No significant differences were observed in S-UA and S-Xa/HX concentrations between the two groups.

Urinary volume and pH
Urinary volume in the SW group was 848 ± 157 mL, whereas 991 ± 252 mL in the SC group. Urinary pH in the SW group was 6.5 ± 0.2, and in the SC group, it was 6.5 ± 0.3. No significant differences in urinary volume and pH were observed between the two groups.

Renal UA metabolic indices
Renal UA metabolic indices are shown in Fig. 1. F UA did not differ significantly between the SW and SC groups (Fig. 1a). U-UA excretion in the SC was significantly higher than in the SW group (SW, 0.45 ± 0.08; SC, 0.52 ± 0.09 mg/kg/h; P < 0.05) (Fig. 1c). Although C UA and FE UA did not differ significantly between the SW and SC groups, C UA and FE UA were slightly higher in the SC group than in the SW group (C UA : SW, 7.76 ± 2.14; SC, 8.75 ± 2.23 mL/min/1.73 m 2 ; P = 0.054; FE UA : SW, 6.08 ± 1.36; SC, 6.64 ± 1.42%; P = 0.060) (Fig.  1b, d).

Renal Xa/HX metabolic indices
Renal Xa/HX metabolic indices are shown in Fig. 2. No significant differences in F Xa/HX , C Xa/HX , and FE Xa/HX were observed between the SW and SC groups (Fig. 2a, b, d). U-Xa/ HX excretion in the SC group was significantly higher than in the SW group (SW, 0.08 ± 0.04; SC, 0.16 ± 0.05 mg/kg/h; P < 0.01) (Fig. 2c).

Discussion
In the present study, we investigated the acute effect of green tea catechins on UA metabolism after alcohol ingestion. We showed that U-UA and U-Xa/HX excretions in the test individuals receiving Shōchū with catechin-rich green tea (SC group) were significantly higher than in those who received Shōchū with water (SW group). C UA and FE UA in the SC group were slightly higher than in the SW group but not significantly so. Hyperuricemia is defined as a S-UA concentration higher than 7.0 mg/dL. The S-UA concentration is tightly regulated by UA reabsorption and excretion in the kidney, and various urate transporters mediate UA reabsorption and excretion [21]. Urate anion transporter 1 (URAT1), which transports UA across the apical membrane of proximal tubule cells, is the major urate reabsorption transporter. The organic anion transporters (OAT) 1 and OAT3 are localized at the basolateral side of the proximal tubular epithelial cells membrane and play an important role in UA excretion. Ethanol ingestion increases the concentration of lactate in the blood [10,11], and lactate reportedly reduces renal excretion of UA by competing for OAT1 and OAT3 with UA [21]. In addition, lactate accelerates the reabsorption of UA in the renal epithelial cells via URAT1 [22]. In contrast, it was reported that green tea polyphenols reduce URAT1 expression and increase OAT1 and OAT3 expressions in the kidney of hyperuricemic mice [13]. In this study, we observed an increase of U-UA excretion in the SC group. Accordingly, our result suggested that green tea catechins might increase U-UA excretion through increasing UA clearance via urate transporters, even though alcohol was ingested.
XO is the key enzyme that produces UA. Previously, green tea polyphenols dose-dependently decreased XO activity in the liver of the hyperuricemic mice [13][14][15]. It was also reported that the consumption of green tea inhibited the increase in plasma XO activity induced by exercise in weight-trained men [23]. In this study, U-Xa/HX excretions were significantly higher in the SC group than in the SW group, indicating that the increase in U-Xa/HX excretion in the SC group might be due to the effect of green tea catechins on XO activity, although we could not measure XO activity directly.
Although we did not directly measure plasma catechin concentrations, several previous studies reported that plasma catechin concentrations are elevated within 1-2 h after intake of green tea [24][25][26]. This could suggest that the promoting effect on UA and Xa/HX excretions in the SC group was due to green tea catechins.
There was a trend toward urinary volume being higher in the SC group than in the SW group (P = 0.073). During the experimental period, all subjects were instructed to drink water, 100 mL/h, and so the total fluid intake was equal. Previous studies suggested that a diuretic response to caffeinecontaining drinks is likely to occur in response to an acute dose of caffeine of about 300 mg or more, being unlikely, however, at doses of about 250 mg or less [27]. In addition, it was reported that a caffeine intake of 6 mg/kg in the form of coffee can induce an acute diuretic effect, whereas 3 mg/kg does not disturb the fluid balance in healthy adults [28]. The catechin-rich green tea used in this study contained 91.4 mg caffeine (equivalent to one cup of coffee), which is a lower dose than used in previous studies [27,28]. Hence, these results suggested that the increase in U-UA excretion in the SC group was due to the effect of green tea catechins and not the effect of caffeine. No significant differences were observed between the two groups regarding S-UA and S-Xa/HX concentrations. In this study, venous blood samples were collected only at 150 min after ingestion of test meal, which is the mid-point of the 5-h urine collection. A previous report showed that plasma concentrations of UA, HX, and Xa peak at 30 or 90 min after beer ingestion (10 mL/kg body weight) [11]. Thus, we were not able to observe the peak values in S-UA and S-Xa/HX concentrations. In the future, postprandial S-UA fluctuation needs to be investigated to clarify the effect of green tea catechins.
Several limitations of our study should be considered. We did not examine the effect of water ingestion, which could have been done by adding a control group. To determine the effect of green tea catechins on UA metabolism, it is necessary to examine the effect of water ingestion and monitor the time course of serum and urinary UA, HX, Xa, and catechin concentrations. Another limitation of this study is that women are SC, Shōchū with catechin-rich green tea. Differences (P < 0.05) between the SW and SC groups were identified using the paired t test or Wilcoxon signed-rank test: an asterisk indicates SW vs. SC not included in this study. It is well known that gout affects men more than women and there is a sex difference in S-UA concentrations [29]; thus, we decided to include only men as the study population. However, S-UA concentrations are known to increase in women after menopause [29], and the effects in women should be evaluated as well. Furthermore, we must take into account the small sample size in this study. Some previous studies reported that FE UA responses were different following the fructose or inosine load between the BMI groups (≥ 25, and < 25 kg/m 2 ), although the BMI groups had similar FE UA at baseline [30,31]. Since the response to green tea catechins may vary among the participants because of their different background characteristics, a larger cohort is clearly required to determine the effect of green tea catechins. Finally, we used catechin-rich green tea, which is equivalent to five cups of regular green tea. Therefore, it is necessary to investigate whether the effect of green tea catechins can be dose-dependently observed for regular green tea.
This study showed significantly higher U-UA and U-Xa/ HX excretions in healthy men receiving Shōchū with catechin-rich green tea than in those receiving Shōchū with water. In conclusion, this study illustrates the potential of green tea catechins to enhance the excretion of UA and Xa/ HX, even when alcohol is ingested simultaneously. It is important to limit excessive alcohol intake for hyperuricemia; however, it is necessary to account for the non-adherence of limiting alcohol intake. This study demonstrates the potential clinical applications of catechin-rich green tea as a dietary component for continued dietary therapy.  Data availability Data that support the findings of this study are available from the corresponding author upon reasonable request.
Code availability Not applicable

Declarations
Disclosures None.
Ethics approval The protocol was approved by the Ethics Committee of the University of Shizuoka.
Consent to participate The purpose of the study was explained to the participants, and the present study was performed after obtaining written informed consent.
Consent for publication Prior to data collection, the purpose of the study was explained to the participants and their informed consent was recorded.