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Levels of uric acid in the intestinal tracts of germfree, gnotobiotic, and conventional mice
Levels of Uric Acid in the Intestinal Tracts of Germfree, Gnotobiotic, and Conventional Mice By JOHN S. COLE, III, ANDRALPH F. WISEMAN The levels of uric acid in various segments of the intestinal tract of germfree, monoassociated, polyassociated, and conventional mice was determined by an enzymatic, spectrophotometric procedure. The amount of uric acid in the intestinal lumen of all mice was found to decrease
as the content moved down the tract. Although uric acid was detected in the distaI portions of the tracts of germfree mice, none was found in the cecum or large intestine of mice that were associated with bacteria. (Metabolism 20: No. 3, March, 278-279, 1971)
S
ORENSEN,l IN AN EXTENSIVE STUDY on the elimination of uric acid in gouty and normal subjects, concluded that the intestinal tract is a primary site of uricolysis in man and that the intestinal bacteria are responsible for the degradation of one third of the uric acid normally turned over each day. He stated: “In most gouty subjects, extrarenal disposal of uric acid is even greater. In some patients with renal insufficiency, enteral uricolysis constitutes the major process of disposal of uric acid.” Our previous studies with rats revealed that the level of ingested uric acid was reduced as it moved down the tract and that uricolytic bacteria may have contributed to this removal.3 To separate the activity of tissue uricolysis from the bacterial activity, these studies were extended to include the germfree and gnotobiotic animal. MATERIALS
AND METHODS
Strain CD 1 germfree mice, COBS “conventional” mice (Charles River Farm, Brookline, Mass.), gnotobiotic (monoassociated, ex-germfree mice, containing a pure culture of a uricolytic Aerobacter aerogenes) and polyassociated animals (ex-germfree, exposed to the total flora of conventional mice) were used in this study. All groups, except the conventional, were maintained in sterilized flexible plastic isolators and were supplied with sterilized diet and water. GLF rat-mouse diet (Charles River Farms) autoclaved for 30 min was fed to all groups of animals. At predetermined periods mice were removed from each group, killed with ether and the intestinal tract removed. Pooled samples of content from the upper and lower small intestine, the cecum and the large intestine of each group were collected. Heart blood was also withdrawn and pooled. The enzymatic procedure of Bare and Wisemad was used for the determination of uric acid levels in the pooled samples. In this assay chromogenic substances are determined by means of a calorimetric procedure before and after the action of uricase. The difference From the Department of Microbiology, University of Kentucky, Lexington, Ky. Received for publication September 29, 1970. This work was supported in part by NIH Grant AM-04311 from the Division of Arthritis and Metabolic Diseases. JOHN S. COLE, III, B.S.: Research Assistant, Department of Microbiology, University of DepartKentucky, Lexington, Ky. RALPH F. WISEMAN, PH.D.: Professor of Microbiology, ment of Microbiology, 278
University
of Kentucky,
Lexington,
Ky.
METABOLISM, VOL. 20, No.
3 (MARCH), 1971
LEVELS
279
OF URIC ACID
Table l.-Average Levels of Uric Acid in Contents from Segments of Intestinal Tract of Germfree, Monoassociated, Poiyassociated and Conventional Mice “g&p” Germfree (23) * Monoassociated with Aerobacter 5 days (14) Polyassociated with “normal” flora 4 days (13) Conventional (25)
Large LowerSmall Intestine Intestine Cecum (mg/g wetcontentper 100g bodyweight)
1.96
0.73
0.37
0.32
1.71
0.63
0
0
1.88 1.79
1.21 1.23
0 0
0 0
* Number of mice in each category. between total color and residual color is a measure of uric acid that has been converted to dlantoin. Weighed portions of the pooled intestinal contents were deproteinized with sodium tungstate and filtered through Whatman No. 40 filter paper prior to assay for uric acid.
RESULTS AND DISCUSSION
The results of this study support our earlier findings that the levels of uric acid decreased as the content moved down the intestinal tract of rodents. Since this event was apparent in germfree mice (Table 1) , it is assumed that uric acid is absorbed from the tract and degraded by uricase located in hepatic microbodies.4 The contribution of the intestinal flora in the removal of uric acid was most apparent in the lower bowels of the groups of mice that were associated with bacteria, since uric acid could not be detected in the contents from the ceca or large intestines. The relatively higher levels of uric acid in the lower small intestine of the polyassociated and the conventional animals suggests that components of the normal flora either synthesize uric acid in the gut or interfere with its absorption from the tract. The “germfree” level in the lower small intestine of the animals monoassociated with the uricolytic Aerobacter aerogenes may be explained by the observation of Figueroa,” who noted this bacterial genus did not possess xanthine dehydrogenase, and that it did not synthesize uric acid in vitro. Preliminary results of the assay for blood uric acid revealed that the germfree mice averaged 0.17 mg/lOO ml serum, whereas the level in the conventional group averaged 0.08 mg/lOO ml. This difference suggests that the removal of uric acid by the intestinal bacteria is reflected in the quantity of uric acid that persists in the blood. REFERENCES 1. Sorensen, L. B.: Role of the intestinal tract in the elimination of uric acid. Arthritis Rheum. 8:694, 1965. 2. Armstrong, P. J., and Wiseman, R. F.: Uric acid degradation by intestinal bacteria of the rat. Bact. Proc. 1962:159, 1962. 3. Bare, L. N., and Wiseman, R. F.: Urate and nonurate chromogens in human serum.
Amer. J. Clin. Path. 40556,
1963.
4. Hruban, Z., and Swift, H.: Uricase: Localization in hepatic microbodies. Science 146:1316, 1964. 5. Figueroa, M. E.: Xanthine dehydrogenase in bacteria, Ph.D. dissertation, University of Kentucky, Lexington, 1967.
as the content moved down the tract. Although uric acid was detected in the distaI portions of the tracts of germfree mice, none was found in the cecum or large intestine of mice that were associated with bacteria. (Metabolism 20: No. 3, March, 278-279, 1971)
S
ORENSEN,l IN AN EXTENSIVE STUDY on the elimination of uric acid in gouty and normal subjects, concluded that the intestinal tract is a primary site of uricolysis in man and that the intestinal bacteria are responsible for the degradation of one third of the uric acid normally turned over each day. He stated: “In most gouty subjects, extrarenal disposal of uric acid is even greater. In some patients with renal insufficiency, enteral uricolysis constitutes the major process of disposal of uric acid.” Our previous studies with rats revealed that the level of ingested uric acid was reduced as it moved down the tract and that uricolytic bacteria may have contributed to this removal.3 To separate the activity of tissue uricolysis from the bacterial activity, these studies were extended to include the germfree and gnotobiotic animal. MATERIALS
AND METHODS
Strain CD 1 germfree mice, COBS “conventional” mice (Charles River Farm, Brookline, Mass.), gnotobiotic (monoassociated, ex-germfree mice, containing a pure culture of a uricolytic Aerobacter aerogenes) and polyassociated animals (ex-germfree, exposed to the total flora of conventional mice) were used in this study. All groups, except the conventional, were maintained in sterilized flexible plastic isolators and were supplied with sterilized diet and water. GLF rat-mouse diet (Charles River Farms) autoclaved for 30 min was fed to all groups of animals. At predetermined periods mice were removed from each group, killed with ether and the intestinal tract removed. Pooled samples of content from the upper and lower small intestine, the cecum and the large intestine of each group were collected. Heart blood was also withdrawn and pooled. The enzymatic procedure of Bare and Wisemad was used for the determination of uric acid levels in the pooled samples. In this assay chromogenic substances are determined by means of a calorimetric procedure before and after the action of uricase. The difference From the Department of Microbiology, University of Kentucky, Lexington, Ky. Received for publication September 29, 1970. This work was supported in part by NIH Grant AM-04311 from the Division of Arthritis and Metabolic Diseases. JOHN S. COLE, III, B.S.: Research Assistant, Department of Microbiology, University of DepartKentucky, Lexington, Ky. RALPH F. WISEMAN, PH.D.: Professor of Microbiology, ment of Microbiology, 278
University
of Kentucky,
Lexington,
Ky.
METABOLISM, VOL. 20, No.
3 (MARCH), 1971
LEVELS
279
OF URIC ACID
Table l.-Average Levels of Uric Acid in Contents from Segments of Intestinal Tract of Germfree, Monoassociated, Poiyassociated and Conventional Mice “g&p” Germfree (23) * Monoassociated with Aerobacter 5 days (14) Polyassociated with “normal” flora 4 days (13) Conventional (25)
Large LowerSmall Intestine Intestine Cecum (mg/g wetcontentper 100g bodyweight)
1.96
0.73
0.37
0.32
1.71
0.63
0
0
1.88 1.79
1.21 1.23
0 0
0 0
* Number of mice in each category. between total color and residual color is a measure of uric acid that has been converted to dlantoin. Weighed portions of the pooled intestinal contents were deproteinized with sodium tungstate and filtered through Whatman No. 40 filter paper prior to assay for uric acid.
RESULTS AND DISCUSSION
The results of this study support our earlier findings that the levels of uric acid decreased as the content moved down the intestinal tract of rodents. Since this event was apparent in germfree mice (Table 1) , it is assumed that uric acid is absorbed from the tract and degraded by uricase located in hepatic microbodies.4 The contribution of the intestinal flora in the removal of uric acid was most apparent in the lower bowels of the groups of mice that were associated with bacteria, since uric acid could not be detected in the contents from the ceca or large intestines. The relatively higher levels of uric acid in the lower small intestine of the polyassociated and the conventional animals suggests that components of the normal flora either synthesize uric acid in the gut or interfere with its absorption from the tract. The “germfree” level in the lower small intestine of the animals monoassociated with the uricolytic Aerobacter aerogenes may be explained by the observation of Figueroa,” who noted this bacterial genus did not possess xanthine dehydrogenase, and that it did not synthesize uric acid in vitro. Preliminary results of the assay for blood uric acid revealed that the germfree mice averaged 0.17 mg/lOO ml serum, whereas the level in the conventional group averaged 0.08 mg/lOO ml. This difference suggests that the removal of uric acid by the intestinal bacteria is reflected in the quantity of uric acid that persists in the blood. REFERENCES 1. Sorensen, L. B.: Role of the intestinal tract in the elimination of uric acid. Arthritis Rheum. 8:694, 1965. 2. Armstrong, P. J., and Wiseman, R. F.: Uric acid degradation by intestinal bacteria of the rat. Bact. Proc. 1962:159, 1962. 3. Bare, L. N., and Wiseman, R. F.: Urate and nonurate chromogens in human serum.
Amer. J. Clin. Path. 40556,
1963.
4. Hruban, Z., and Swift, H.: Uricase: Localization in hepatic microbodies. Science 146:1316, 1964. 5. Figueroa, M. E.: Xanthine dehydrogenase in bacteria, Ph.D. dissertation, University of Kentucky, Lexington, 1967.