Lactobacillus reuteri TSR332 and Lactobacillus fermentum TSF331 stabilize serum uric acid levels and prevent hyperuricemia in rats

Probiotic cultivation and analysis of purine nucleoside assimilation in vitro

Nineteen probiotic strains, including TSR332 (Lactobacillus reuteri); TSF331, L-18, and L-319 (Lactobacillus fermentum); La-322 (Lactobacillus amylovorus); TSP05, L-29, and L-335 (Lactobacillus plantarum); L-120 (Lactobacillus paracasei); L-87 (Lactococcus lactis subsp. lactis); L-39 (Bifidobacterium animalis subsp. lactis); L-85 and L-134 (Lactobacillus rhamnosus); L-243 (Streptococcus thermophilus); L-69 (Bifidobacterium animalis subsp. lactis); L-291 (Lactobacillus acidophilus); L-9 (Lactobacillus casei subsp. casei); Bv-889 (Bifidobacterium breve); and L-2 (Lactobacillus gasseri), were provided by Glac Biotech Co., Ltd. (Tainan, Taiwan). TSR332 and TSF331 were isolated from human gut, and TSP05 was isolated from kimchi. Four probiotic strains, including BCRC14045 and BCRC14060 (Bifidobacterium breve); and BCRC14619 and BCRC17616 (Lactobacillus gasseri), were purchased from the Bioresource Collection and Research Center, Hsinchu City, Taiwan. One probiotic strain, LGG (Lactobacillus rhamnosus), was obtained from Chr. Hansen Inc. (Milwaukee, WI). The probiotics were cultured in de Man, Rogosa & Sharpe broth containing 0.05% cysteine for 20 h.

To measure purine assimilation, 10⁹ probiotic cells were isolated and incubated in one mL of trisodium phosphate solution (0.1 M) containing 1.25 mM inosine or guanosine for 30 min. Then, 0.9 ml of supernatant was collected and mixed with 0.1 mL of perchloric acid to stop purine degradation. Inosine, guanosine, hypoxanthine, and guanine levels in the supernatant were analyzed using high-pressure liquid chromatography (HPLC). The degradation rate of nucleosides was calculated as (standard _{total area of nucleosides} – sample _{total area of nucleosides}) / standard _{total area of nucleosides} (%). Purine base generation was calculated as sample _{total area of base} / standard _{total area of nucleosides}.

HPLC

Purine and purine bases were analyzed using an Altus HPLC instrument equipped with a photodiode array detector (PerkinElmer, MA, USA). The analysis column was a Kinetex 2.6 µm F5 100A (150 × 3 mm, Phenomenex, CA, USA). The mobile phase solutions were methanol and H₂O. The samples (3 µL) were injected into the HPLC column at 28 °C, with a flow rate of 0.25 mL/min. The optical density at 254 nm was measured in the eluted samples. The results were analyzed using the Empower 3 software (Waters, MA, USA).

Animal treatment

The experiments were carried out according to regulations controlled by the Animal Research Committee of HKU; all experimental protocols were approved by the Ethical Committee of the Hungkuang University (approved affidavit No.: HK-P-10512). Animal studies were carried out in the Department of Food Science and Technology, Hung Kuang University. Thirty-day old male Wistar rats were purchased from BioLASCO Taiwan Co., Ltd and were housed one rat per cage at the Laboratory Animal Center, Hungkuang University (HKU). The animals arrived with initial weights of approximately 100 g and were given free access to sterilized water and food. The rats were housed under a 12-h light/dark cycle, at 22 ± 2 °C, and 62% ± 5% humidity. The rats were familiarized with human handling and the laboratory one week prior to the start of the experiments. All animals were healthy, and no signs of pain or distress were observed. No euthanasia was needed during the experiments. All animals were anesthetized using Zoletil/Xylazine before cervical dislocation. Animal experiments and protocols were in compliance with the Laboratory Animal Care and Use Guidelines published by the Taiwanese government.

Hyperuricemia induction and experimental setup

To elicit hyperuricemia, the rats were fed a high-purine diet containing 87 g yeast extract (Sigma, St. Louis, MO) and 1.5 g ribonucleic acid from torula yeast (R6625; Sigma) to induce hyperuricemia. Subsequently,rats were injected intraperitoneally with potassium oxonate (0.35 mg/100 g body weight/day) dissolved in carboxymethylcellulose sodium solution (3 g/L). Bodyweight, UAs, and creatinine levels in the hyperuricemia model rats were analyzed at selected time points.

For the control group (C), rats received normal diets (AIN-93G) and oxonate injections. For the symptom control group (HP), rats received high-purine diets and oxonate injections. For probiotic groups, rats receive high-purine diets, probiotics supplements, and oxonate injections. The probiotic strains were given via oral 1,000-µL gavages containing 10⁹ CFU/kg/day of each probiotic strain in 0.85% NaCl. Eight rats were examined per group.

To investigate the effect of TSF331, TSR332, and La322 in hyperuricemia rats, experiments were carried out for 8 days, as shown in Fig. 1A. Blood samples were collected on days 0 and 8.

To investigate the preventive and therapeutic effects of TSR332, experiments were carried out, as shown in Fig. 2A. During days 1 through 6, TSR332 was administered daily in two groups: the prevention and combination groups. On day 7, all groups were injected with potassium oxonate for another 8 days. During days 7 through 14, TSR332 supplementation was stopped in the prevention group and was continued in the combination group. The therapeutic group was treated with TSR332, simultaneously with potassium oxonate injection, from day 7 to 14. Rats in the symptom control (HP) were given a high-purine diet from day 1 to 14 and a potassium oxonate injection from day 7 to 14. Blood samples were collected on days 0, 7, and 14.

To investigate the therapeutic effect of TSR332 in rats with severe hyperuricemia, experiments were carried out for 14 days, as shown in Fig. 3A. Body weights were measured, and blood samples were collected on days 0, 7, and 14.

Evaluation of body weight, creatinine, and UA

The animal body weight was measured, and blood samples were collected for UA and creatinine analyses on days 0, 7, and 14. One milliliter of blood was collected from the tail vein of each animal and the serum was obtained by centrifugation at 1350 × g for 5 min. UA levels were determined using UA stript#25 and an EasyTouch GCU multi-function monitoring system (Bioptik Technology, Inc., Miaoli County, TW). Creatinine levels were determined using LabAssay#290-65901 (Wako Chemicals Inc., VA, USA) and an Automated Clinical Chemistry Analyzer (FUJI DRI-CHEM 4000i; Fuji, Japan).

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. Data are presented as the mean ± standard deviation (SD) obtained from eight animals. Differences were evaluated using one-way ANOVA followed by Tukey’s range test for multiple comparisons. A p < 0.05 was considered statistically significant.

L. reuteri TSR332 and L. fermentum TSF331 rapidly metabolize purine nucleosides, without the production of UAs

To screen potential probiotic strains, 24 strains (Table 1) were tested for inosine and guanosine assimilation abilities based on HPLC detection. Compounds detected in inosine assimilation were inosine, xanthine, hypoxanthine, and UA. Compounds detected in guanosine assimilation were guanosine, xanthine, guanine, and UA. Purine degradation for inosine and guanosine by strain TSR332 (Figs. 4A, 4C) showed an apparent decrease in purines and an increase in purine derivatives when compared to that of standard solutions (Figs. 4B, 4D). HPLC results for all strains are summarized in Table 2. The majority of the 24 tested strains exhibited purine assimilation, with an average assimilation rate of 25.72% for inosine and of 20.54% for guanosine. Among them, strains TSR332 and TSF331 were found to metabolize purine nucleosides at rates higher than 50% within 30 min. In particular, TSR332 showed the highest rates of 90.05% and 78.02% for inosine and guanosine, respectively.

To identify downstream degradation products produced by the probiotic strains, the same solutions used in the above tests were further analyzed for purine metabolites by HPLC. The results are summarized in Table 2. Hypoxanthine and guanine were the two main components of the metabolites, and UA was not detected. TSR332 and TSF331 displayed the highest purine-metabolizing rates within 30 min. In a 1.25 mM inosine solution, 1.126 mM and 0.743 mM of inosine was degraded, and 0.754 mM and 0.497 mM of hypoxanthine was produced by TSR332 and TSF331, respectively. In a 1.25 mM guanosine solution, 0.975 mM and 0.641 mM of guanosine was degraded, and 0.083 mM and 0.327 mM of guanine was produced by TSR332 and TSF331, respectively (Table 2).

Inosine and guanosine assimilation rates were compared side by side for the 24 strains. The purine assimilation ability varied widely, from 2.15% to 90.05%, among the strains (Fig. 5, Table S4). Inosine and guanosine assimilation rates for L-85 were very low (3.98% and 5.77%, respectively). Moderate inosine and guanosine assimilation rates were observed for La-322 (37.17% and 17.85%, respectively). Like TSR332, TSF331 exhibited high inosine and guanosine assimilation rates, of 59.43% and 51.26%, respectively (Fig. 5). The two outstanding stains, TSR332 and TSF331, together with one moderately performing strain, La322, were selected for the animal study.

L. reuteri TSR332 and L. fermentum TSF331 stabilize the level of UA in hyperuricemia model rats

To preliminarily examine the effects of TSR332, TSF331, and La322 on serum UA levels in rats, hyperuricemia was induced by intraperitoneal injection of potassium oxonate and a high-purine diet. Probiotic strains were supplemented to hyperuricemia rats for 8 days (Fig. 1). The outline of this study is presented in Fig. 1A. The blank control group was fed a regular diet, and 4 other groups were fed a high-purine diet to increase the purine uptake from food. One hyperuricemia group served as the symptom control (HP), and three of the hyperuricemia groups were supplemented with probiotic strains daily for 8 days. Blood samples were taken on days 0 and 8. The results showed that hyperuricemia was induced successfully as soon as potassium oxonate was injected, and serum UA levels were significantly (^##p = 0.00598) higher in the HP group (4.99 mg/dL) than in the control group (1.86 mg/dL) (Fig. 1B). Supplementation of TSF331 and TSR332 significantly (^∗∗p = 0.00811 and ^∗∗p = 0.00223) reduced the UA levels to 3.57 mg/dL and 1.92 mg/dL, respectively. Remarkably, TSR332 reduced the UA level to a greater extent than that by TSF331, whereas La322 reduced UA to a level (4.62 mg/dL) comparable to that in the HP group (Fig. 1B, Table S1).

L. reuteri TSR332 displays preventive and therapeutic effects in stabilizing the serum UA level in hyperuricemia model rats

To investigate the preventive and therapeutic effects of TSR332 in rats further, more treatment schemes were evaluated and longer treatment periods were used (Fig. 2). The outline of this animal study was presented as Fig. 2A. The blank control group was fed a regular diet, and 4 other groups were fed a high-purine diet to increase purine uptake from food. The symptom was induced by oxonate injection on day 7-14. One hyperuricemia group served as the symptom control (HP), and three other hyperuricemia groups were supplemented with probiotic strains for the designated periods (pretreatment on days 1–7, treatment on days 7–14, or probiotics on days 1–14). Blood serum was collected and tested for UA on days 0, 7, and 14. On day 0, serum UA levels were similar in all groups. After the beginning of potassium oxonate injection on day 7–14, UA levels on days 7 and 14 in the symptom control (HP) group (3.26 mg/dL; ^##p = 0.00772 and 3.64 mg/dL; ^##p = 0.00307, respectively) increased significantly as compared to the levels in the blank control group (1.77 mg/dL and 1.60 mg/dL, respectively) (Fig. 2B). UA levels were significantly lower in the pretreatment group than in the HP group on both day 7 and 14, and a more dramatic reduction was seen on day 7 (1.95 mg/dL, ^∗∗∗p = 0.00088) than on day 14 (2.43 mg/dL, ^∗∗p = 0.00921). UA levels were slightly lower in the treatment group (2.74 mg/dL) than in the HP group (3.26 mg/dL) on day 7 and were significantly lower on day 14 (2.26 mg/dL vs. 3.64 mg/dL, ^∗∗p = 0.00815). On day 7, in the probiotics group, the UA levels (2.40 mg/dL) were significantly (^∗p = 0.03084) lower than those in the HP group (3.26 mg/dL), and the difference increased further (2.38 mg/dL vs. 3.64 mg/dL, ^∗∗p = 0.00721) on day 14 (Fig. 2B, Table S2).

L. reuteri TSR332 stabilizes the UA level in acute hyperuricemia rat model

To investigate the therapeutic effects of TSR332 on acute hyperuricemic rats, hyperuricemia was induced using a high-purine diet and potassium oxonate injection for 14 days straight. Treatments were similar to those used for the preliminary test, except for the duration (Fig. 3A). The blank control group was fed a regular diet, and two other groups were fed a high-purine diet to increase purine uptake from food. One hyperuricemia group served as the symptom control (HP) and another hyperuricemia group was supplemented with probiotic TSR332 daily for 14 days. On day 0, UA levels were similar in all groups (Fig. 3B). The high-purine diet and potassium oxonate injection consistently and significantly induced high UA levels in the HP group on days 7 and 14 (3.73 mg/dL; ^##p = 0.00705 and 3.60 mg/dL; p = 0.00519, respectively). When compared with the HP group, supplementation of TSR332 significantly decreased UA levels on days 7 and 14 (2.38 mg/dL; ^∗∗∗p = 0.00091 and 2.35 mg/dL; ^∗∗p <0.00127, respectively; Fig. 3B, Table S3).

Body weights increased over time in the control group (96.63 g to 214.37 g) and were significantly lower in the HP group (125.20 g; ^#p = 0.0356 and 160.07 g; ^##p = 0.00664, respectively) than in the control group on days 7 and 14, whereas no significant difference was detected between HP group and the TSR332 group (131.38 g and 170.13 g, respectively; Fig. 3C, Table S3). To evaluate the effect of TSR332 on kidney function, blood creatinine levels were analyzed and were found to vary between 0.18–0.35 mg/dL. Although some differences were seen among the groups, they were not significant (Fig. 3D, Table S3).