32[Noda Institute for Scientific Research GRANT]2006 Research Grant
Outline of Research Result

Analysis of Microbial Purine Metabolism and Its Applicationfor Hyperuricemia Prevention
Jun OGAWA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University

Research aims
Hyperuricemia is a disease, which results from the overproduction and/or underexcretion of uric acid and is greatly
influenced by a high dietary intake of purine. It is usuallyan asymptomatic condition, which is hypothesized to play arole in cardiovascular disease and hypertension. Some hyperuricemic individuals develop gout, a common disease
with a worldwide distribution and are mainly caused bydeposition of monosodium urate crystals in joints and othertissues as a result of extracellular urate supersaturation. Although most uric acid is derived from the metabolism ofendogenous purine, eating foods rich in purines contributes
to the total pool of uric acid. Consumption of meat, seafood
and alcoholic beverages in moderation and attention to foodportion size is important. Low purine diet, moderation in
the consumption of not only beer but also other forms of alcohol is essential. But traditional low-purine diets, once a
mainstay of hyperuricemia management, are difficult forpatients to adhere to because it is not easy to keep the nutritional balance of their diets.
We attempted to design a new prophylaxis for hyperuricemia using probiotic effects of microorganisms. The
term probiotic refers to live microorganisms that survive
passage through the gastrointestinal tract and have beneficial effects on the host. We assumed that probiotic bacteria
administered orally could help degrading purine and purine
metabolites derived from food in human intestine, whichlead to lowering the elevated serum uric acid level. In thisreport, we first described about the screening of microorganisms which catalyze inosine and guanosine degradationand evaluated their effects on serum uric acid level reduction. Furthermore, through the analysis of purine metabolism, mechanism of the probiotic effects of the potential
strains was elucidated.
Methods
Lactic acid bacteria (267 strains in total), including Bifidobacterium, Lactobacillus, Enterococcus, Leuconostoc,
and Pediococcus species, were cultivated anaerobically inMRS medium and their washed cells were prepared. The
washed cells were added to the reaction mixture containing
1.25 mM inosine, 1.25 mM guanosine, and 0.1 M potassium

phosphate buffer (pH 7.0) and incubated anaerobically 2 hat 37°C. After the reaction, the concentration of inosine andguanosine was monitored and the strains showed higher reduction were selected.
The effects of selected strains on the serum uric acidlevel were investigated using the rats pretreated with theuricase inhibitor potassium oxonate as animal models for
hyperuricemia. The non-treated group was fed the diets
containing 0.25% oxonate. The control group was fed thediets containing 0.25% oxonate and 1% RNA as purine
source. The test group was fed the diets containing 0.25%
oxonate and 1% RNA together with washed cells of lactic
acid bacteria (1.0109 CFU). After 2, 5, and 8 days of feeding, the serum was collected and their uric acid level wasevaluated.
As to the strains showing serum uric acid level reduction,
their purine metabolism was analyzed. These strains were
cultivated in MRS medium under anaerobic conditions at28°C for 48 h. 2 ml of the reaction mixture comprising 100mM potassium phosphate buffer (pH 7.0) and 1.25 mMAMP, 1.25 mM IMP, 1.25 mM GMP, 1.25 mM adenosine,
1.25 mM inosine, 1.25 mM guanosine, 1.25 mM adenine,
1.25 mM hypoxanthine, 1.25 mM guanine, 1.25 mM xanthine, or 1.25 mM uric acid were added into the test tube
containing the wet cells from 3 ml culture broth and incubated anaerobically at 37°C for 2 h. After the incubation,
the purine contents in the supernatants were analyzed.
Results
Microorganisms were screened for their ability to degrade purine. As the result of screening in Bifidobacterium,Lactobacillus, Enterococcus, Leuconostoc and Pediococcus
species, 13 strains of bacteria, such as L. mali, L. vaccinostercus, L. brevis, L. fermentum, L. homohiochii, and L. pentosus, were selected as good catalysts for degradation of inosine and guanosine. Among these 13 strains, 11 strains
were isolated from fermented food and good for probiotic
purposes. As to these 11 strains, the effects on the serum
uric acid level were investigated using the rats pretreatedwith the uricase inhibitor, potassium oxonate, as animalmodels for hyperuricemia. During the testing term, no significant difference in weight and dietary intake was observed with each group. The serum uric acid level of con-33trol group was increased until 5 days and reached maximum. On the other hand, the test group with lactic acid bacteria intake showed repressing effects on serum uric acidlevel elevation. The ability to lower elevated level of serum
uric acid was found in L. fermentum ONRIC b0185, L. fermentum ONRIC b0195, and L. pentosus ONRIC b0223(Fig. 1).
To elucidate the mechanism to lower the elevated serum
uric acid levels, purine metabolism, i.e., the degradation ofIMP, AMP and GMP and their metabolites, was evaluatedfor L. fermentum ONRIC b0185, L. fermentum ONRICb0195, and L. pentosus ONRIC b0223. L. fermentum
ONRIC b0185 converted adenosine to adenine, inosine tohypoxanthine, guanosine to guanine, and guanine to xanthine. This strain strongly degraded inosine to hypoxanthine, and guanosine to guanine (Fig. 2). L. fermentum
ONRIC b0195 converted adenosine to adenine, inosine tohypoxanthine, guanosine to guanine and xanthine. In a reaction with guanine, guanine was not degraded to xanthinein 2 h. On the other hand, this strain converted guanine toxanthine in a reaction with guanosine (Fig. 2). L. pentosus
ONRIC b0223 converted adenosine to adenine, inosine tohypoxanthine, and guanosine to guanine. This strainstrongly degraded inosine to hypoxanthine (Fig. 2). All ofthese strains well converted nucleosides to purine bases, indicating that they have high nucleosidase activity.

Fig. 1. Results of hyperuricemia model rat experiments. Black triangle; serum uric acid level of non-treaded group with oxonate containing diets, bluesquare; serum uric acid level of control group with oxonate and RNA containing diets, red circle; serum uric acid level of test group with oxonate, RNA, andlactic acid bacteria containing diets.

Fig. 2. Purine degradation pathway of lactic acid bacteria. Dot-arrow; no or trace activity, solid arrow; significant activity. The boldness of the arrow represents the level of activity.

34Fig. 3.

Proposed mechanism of lowering serum uric acid level by lactic acid bacteria.

Conclusion
The ability to lower elevated level of serum uric acid wasfound in L. fermentum ONRIC b0185, L. fermentum
ONRIC b0195, and L. pentosus ONRIC b0223. Thesestrains showed high nucleosidase activity converting nucleoside to corresponding nucleobase.
The previous research about nucleoside transport andmetabolism in isolated rat jejunum reported that luminal inosine and hypoxanthine increased the serosal uric acid concentration significantly and that the nucleoside gave a significantly greater rate of serosal uric acid appearance thanthe base. The research also reported that luminal guanosineand guanine increased the serosal uric acid concentration
significantly and that the serosal uric acid appeared signifi-

cantly faster from the nucleoside than it did from the base.
Taking these facts into account, the result of the analysis ofpurine metabolism of these three strains shows that thesestrains converted purine nucleosides to purine bases, lesseasy form in intestine to be absorbed into serosal side. This
conversion enabled these strains to lower the elevatedserum uric acid level in animals. This result suggested that
this lowering effect might be beneficial in the treatment ofpatients with hyperuricemia using probiotics.
References
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