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Studies in Urolithiasis: IV. Urinary Glucuronosides and Calcium Phosphate
Tull JOURNAL OF UBOLOGT
Vol. 74, No. 4, October 1956 Printed in U.S.A.
STUDIES IN UROLITHIASIS: IV. URINARY GLUCURONOSIDES AND CALCIUM PHOSPHATE EDWIN L. PRIEN
AND
BURNHAM S. WALKER
From Boston University School of Medicine, Boston, Mass.
The actual cause of calculus formation in the urinary tract remains unknown in the great majority of cases. Hypersecretion of crystalloids of low solubility, or their decrease in solubility because of extremes of pH, are mechanisms and not causes. Under these circumstances, attempts to prevent recurrence of urinary calculus are based upon prevention of excessive supersaturation1 • 2 of the urine by substances which might precipitate to form stone. It is assumed, of course, that the well-recognized diagnostic and therapeutic measures have been carried out. These would include surgical removal of parathyroid adenomas and urinary tract obstructions, treatment of infections, and mobilization of recumbent patients. In a certain small number of cases a cure will be obtained by these therapies; there will be no more stone. However, in the majority of cases further treatment must consist of attempting to lower the urinary saturation of the offending crystalloids, by decreasing their output, increasing their solubility in the urine, or both. For calcium-containing stone, and this includes all crystalline calculi except uric acid and cystine, we may decrease the calcium or phosphorus in the diet or prevent the absorption of phosphorus by administration of the aluminum gels. We may also attempt to increase the solubility of calcium phosphate and magnesium ammonium phosphate by acidification therapy. The solubility of calcium oxalate is uninfluenced by changes in pH over the normal range. If a urea-splitting infection is present, the urine may not be rendered acid by any therapy other than eradication of the infection. For uric acid and cystine stone only alkalinization of the urine is of value. Hyaluronidase therapy 3 based on a theory of doubtful foundation 4 has also been tried with equivocal results. 6 In general, the prevention of recurrence of calcium-containing calculi by the various suggested regimens has not been highly successful; many physicians believe that such regimens are useless and prescribe only a large fluid intake. Our results have not been entirely unfavorable with some of these regimens, probably because of an intense interest in the subject. Their limitations have stimulated a search for some other way of decreasing The research reported in this paper was supported by a grant-in-aid from the American Urological Research Foundation, Inc. Read at annual meeting, American Urological Association, Los Angeles, Calif. May 19, 1955. 1 Meyer, J.: tl'ber die Ausfii.llung von Sedimenten und die Bildung von Konkrementen in den Harnwegen . Ztschr. f . klin. Med., 111: 613---687, 1929. 2 Prien, E. L. : Studies in urolithiasis: III. Physicochemical principles in stone formation and prevention . J. Urol., 73: 627-652, 1955. 3 Butt, A. J., Seifter, J . and Hauser, E . A.: Some recent advances in etiolo~y and treatment of kidney stones . Ohio State Med . J., pp . 21-24, 1955. 4 Smiddy, F. G.: Hyaluronidase and urinary stone. Proc. Roy. Soc. Med., 47: 807-809, 1954. 5 Prien, E. L . : Use of hyaluronidase to prevent urinary calculi. Report of a case in which stone recurrence was facilitated by hyaluronidase therapy. J .A.M .A., 154: 744-747, 1954. 440
STUDil:B IN UROLITHIABIS
441
the saturation of the urine of patients with recurrent calcium stone. We have reasoned that the ideal therapy would be one which did not interfere with normal homeostatic mechanisms by interfering with the absorption of calcium or phosphorus from the bowel, was not dependent on the maintenance of an acid urine, would be effective in an alkaline urine, did not require the daily ingestion of an unpleasant medication, could be taken orally in the form of tablets or capsules, was harmless in proper dosage, and not an excessive financial burden. GLUCURONOBIDEB
We learn from the work of others that increasing the concentration of glucuronosides in the urine should increase the solubility of calcium salts. Glucuronosides contain glucuronic acid, and are formed by the conjugation of this substance with any one of
1
HOH7 HCOH
I
HOCH
O Glucuronic acid
H6:JH H6
I
COOH many phenols and alcohols, of either metabolic or extraneous origin. Glucuronic acid conjugation is the most common of the chemical changes usually designated by the term "detoxication," in which a foreign compound in the body is changed to a derivative which is excreted in the urine. The glucuronic acid which is necessary for the biosynthesis of urinary glucuronosides is formed in the body from 3-carbon intermediates originating from glucose or glycerol. 8 An immediate metabolic precursor of urinary glucuronosides is uridine diphosphate glucuronic acid. This substance reacts with a suitable phenolic or alcoholic acceptor forming the glucuronoside with liberation of uridine and inorganic phosphate.7 Glucuronosides are normal urinary components. Free glucuronic acid is not found in the urine unless the substance itself or its lactone has been fed. Even under these circumstances about 80 per cent of a 5 gm. dose is not recovered in the urine. 8 Glucuronic acid combined as urinary glucuronosides may vary from 0.3 to 0.75 gm. per 24 hours, as reported for a single individual. 9 Somewhat larger ranges have been reported when groups have been studied. 10 8 Doerschuk, A. P.: Radiotracer studies of the biosynthesis of conjugated o-glucuronic acid. J. Biol. Chem., 195: 855-859, 1952. 7 Storey, I. D. E . and Dutton, G. J .: Uridine compounds in glucuronic acid metabolism. 2. The isolation and structure of 'uridine-diphosphate-glucuronic acid'. Biochem. J., 59:
279-288, 1955. 8 Ormsby, A. A.: The metabolism of glucuronic acid in man. Texas Rep. Biol. & Med., 10: 66-72, 1952. 9 Fishman, W. H ., Smith, M., Thompson, D ., Bonner, C. D., Kasdon, S. C. and Homburger, F.: Investigation of glucuronic acid metabolism in human subjects. J. Clinical Investig., 30: 685-696, 1951. 10 Artz, N- . E . and Osman, E. M.: Biochemistry of Glucuronic Acid. New York: Academic Press, Inc., 1950.
442
EDWIN L. PRIEN AND BURNHAM S. WALKER
After surveying the large number of compounds known to be excreted as urinary glucuronosides11 we decided that salicylate best met our specifications of easy availability and low toxicity. Salicylate is excreted in the urine with from 15 to 40 per cent of the total excretion conjugated with glucuronic acid. 12 Salicylate is also excreted partly as a glycine conjugate, salicyluric acid, partly as a metabolic product, gentisic acid, and partly as unchanged salicylate. 13 Acetylsalicylic acid yields similar excretory compounds. 14 Salicylamide is excreted as the glucuronoside, as the sulphate, and as the glucuronoside of its metabolic product gentisamide. 16 In our clinical studies to be summarized later, acetylsalicylic acid was used in the majority of cases to increase the urinary output of glucuronosides. In a few instances salicylamide was used. In neutral or alkaline aqueous media, calcium carbonate and calcium phosphate are insoluble. The presence of glucuronides in solution increases the solubility of these substances. 16 Complex glucuronosides have a greater solvent effect than simple glucuronic acid.17 The precipitation of calcium salts in urine as a result of bacterial decomposition of soluble complexes has been observed. 18 Urinary glucuronosides have been observed to be destroyed by Staphylococcus albus19 and many other bacterial species may be presumed to act in a similar manner. The maximum increase in solubility of calcium phosphate brought about by the presence of a glucurononoside has been reported at 43 per cent. 20 THERAPY
For the last 20 months we have treated patients with recurrent calcium-containing calculi by using a daily dosage of 2 gm. of acetylsalicylic acid divided into 3 doses a day. The doses were put in capsules, the aspirin having first been incorporated as a powder into a mixture of harmless excipients for the purpose of disguising it. All patients who received this medication were informed that it was experimental and harmless, but the name of the drug was not divulged. We wished to impress upon the patients the importance of regular use of the drug, and we feared that the drug was too familiar to be impressive. The patients were also instructed to drink fluids, other than milk, liberally. Moderation was urged 11 Teague, R. S.: The conjugates of o-Glucuronic acid of animal origin. Advances in Carbohydrate Chemistry, 9: 185-246, 1954. 12 Alpen, E. C., Mandel, H . G., Rodwell, V. W. and Smith, P. K.: The metabolism of C" carboxyl salicylic acid in the dog and in man. J. Pharmacol., 102: 150-155, 1951. 13 Kapp, E. M . and Coburn, A. F.: Urinary metabolites of sodium salicylate. J. Biol. Chem., 146: 549- 565, 1942. 14 Smith, M. J . H . : Some recent advances in the pharmacology of salicylates. J. Pharm. Pharmacol., 6: 81- 93, 1953. 16 Becher, A., Mikseh, J., Rambacher, P. and Schafer, A.: 'Ober das Verhalten des Salicylamid in Stoffwechsl des Menschen. Klin. Wochschr., 30: 913-918, 1952. 16 Mandi, I., Grauer, A. and Neuberg, C.: Solubilization of insoluble matter in nature. II. The part played by salts of organic and inorganic acids occurring in nature . Biochemica et Biophysica Acta, 10: 540-569, 1953. 17 Neuberg, C. and Grauer, A.: Enzymic cleavage of conjugated glucuronic acids in relation to the problems of mineralysis. Enzymologia, 16: 115-121, 1952. 18 Flocks, R.H.: Nature of urinary calcium: its role in calcium urolithiasis. J. Urol., 64: 633- 637, 1950. 19 Barber, M., Brooksbank, B. W. L . and Haslewood, G. A. D. : Destruction of urinary glucuronide by bacteria. Nature, 162: 701-702, 1948. 2 ° Cessi, C.: La solubilizzazione del fosfato tricalcico durante la sintesi di un glucuronide studiata con fosforo radioattivo. Boll. soc. ital. biol. sper., 30: 629-632, 1954.
STUDIES IN UROLITHIASIS
443
in use of milk or cream with coffee or cereal, and in the use of ice creams, dried fruits, nuts, chocolates, and sea foods. Butter was allowed freely but cheese was forbidden. This high fluid, low calcium diet, together with the aspirin capsules, constituted the entire therapeutic program for the majority of our patients. The few exceptions will be noted in the following section. RESULTS
The treatment just outlined has been prescribed for patients who have had frequently recurring calcium-containing calculi and for a small series of 6 patients in whom alkaline encrusting cystitis developed in the presence of inlying urethral catheters. The catheters became obstructed by calcium phosphate and magnesium ammonium phosphate deposits, requiring that the catheters be changed at intervals of 1 or 2 weeks. In all six of these patients there was satisfactory reduction of the encrustation.* None of these patients was uremic. We have made no trials of aspirin therapy in uremic patients and we do not recommend it since there is danger from failure of renal excretion of the drug. Our stone patients were ambulatory and were selected for this therapy on the basis of the history of recent recurrences of calcium-containing stone. In this report we will consider only 19 of these patients, this being the number who have received the therapy continuously for 1 year or more. We plan to report in the future on the larger series of patients when they have been under treatment long enough for the results to be reasonably conclusive. Of these 19 patients, all but two have a history of at least three separate recurrences. One of these patients had passed over a hundred stones in 14 years. Twelve of these patients had collectively required twenty-three major operations on kidneys or ureters for removal of calculi. Six of this group had presumably permanent scarring of the upper urinary tract, ureteral stricture, residual hydronephrosis, or gross deformity of the pelves or calyces. None of these patients had gone a year without a recurrence of a urinary calculus and some were carrying calculi at the time. All of these patients had pyelographic studies before the treatment was started, supplemented by measurements of serum calcium, serum inorganic phosphorus, and nonprotein nitrogen. Also, two 24-hour urine specimens were collected and studied on each patient while he was on the high-fluid, low-calcium diet, but before aspirin was given; these constituted the control or pre-treatment specimens. The measurements carried out included volume, pH, calcium, inorganic phosphate, magnesium, and total glucuronic acid. 9 This last mentioned analysis includes of course the normal urinary glucuronosides plus the extra glucuronosides produced as a result of the administration of salicylate, all expressed as glucuronic acid. These same analyses were repeated on 24-hour specimens collected after 2 weeks of treatment and at intervals thereafter, at least once in 3
* We have subsequently encountered several bedridden patients in whom encrustation of catheters was not prevented by salicylate therapy. It is probable that the glucuronide solubilizing mechanism may be inadequate to prevent precipitation of salts when present in excessive amounts in very alkaline urine. The remarkable effect of pH on solubility of the phosphate salts in urine is emphasized in the previous paper in this series (J. Urol., 73: 640, 1955); normal urine is 22 times supersaturated with calcium phosphate at pH 8.
444
EDWIN L. PRIEN AND BURNHAM 8. WALKER
months. Some of the patients who lived at a distance were followed under the care of their local physicians who supervised the collection of 24-hour specimens, sterilized them, and sent us aliquot portions by mail. The output of total glucuronic acid ranged from 0.2 to 0.9 gm. per 24 hours in the patients who had not started aspirin therapy. While the patients were receiving aspirin the outputs ranged from 0.6 to 2.2 gm. per 24 hours. It was almost always possible to double the output and frequently to triple or quadruple it. Following a year or more of treatment all of these patients had plain urograms of the kidneys, ureters and bladder, excretory urograms, or both to determine if existing calculi had grown or new stones had appeared. These films were compared with those obtained before drug therapy was started. In 17 of these 19 patients, no new calculi appeared, nor was there increase in size or density of calculi present at the start. In one of the other two patients, there was a slight increase in size of his small renal calculi after 18 months under treatment. In this particular patient we were never able to achieve the usual increase in the output of glucuronic acid. We have no reason to think that this patient did not take the drug faithfully. It seems more reasonable to conclude that he is a poor glucuronoside producer or that there was excessive destruction of glucuronosides by bacterial action. The one remaining patient was a complete failure, although his glucuronoside production was excellent. His calculi became larger and additional calculi made their appearance. This patient differs from the others in the series in respect to the composition of his calculi. The other patients had produced stones composed chiefly of the usual type of calcium phosphate, or apatite stone. 21 We may call this basic calcium phosphate because it contains hydroxyl (OH) radicles. The patient in whom the therapy failed formed calculi composed of acid calcium phosphate (CaHP04). This compound occurs in less than 2 per cent of human renal calculi. Out of this group of 19 patients, 16 had infected urines when treatment was started. The organisms cultured included Pseudomonas aeruginosa, Proteus vulgaris, Micrococcus pyogenes, E. coli, Aerobacter aerogenes, Lactobacilli and diptheroids. Most of these infections had been of long standing and the patients had acquired strains resistant to the common antibacterial agents. One patient's urine was cleared of infection by supplemental antibacterial therapy during the period of treatment. Several of the other patients with infected urines became clear of the infection without additional therapy. In this group and in the larger group not presently being reported, there have been relatively few undesirable side effects of aspirin therapy. Signs of salicylate poisoning have been watched for, but for the most part have not appeared. Only one patient reported a transient ringing in the ears. Prothrombin time was determined and found normal in several patients with borderline renal function. No instance of granulocytopenia was noted. Several patients have reported 11 Prien, E. L. and Frondel, C.: Studies in urolithiasis. I. The composition of urinary calculi. J. Urol., 67: 949-991, 1947.
STUDIES IN UROLITHIASI8
445
gastric discomfort or heartburn, and two of these have been carried successfully on salicylamide, with the production of satisfactory outputs of urinary glucuronosides. Therapy had to be discontinued in one patient on account of an exacerbation of a duodenal ulcer which had previously caused no symptoms. DISCUSSION
This incomplete and preliminary report on a small group of patients is published at this time with the simple purpose of suggesting a new line of therapeutic approach to the problem of recurrent calcium-containing stones. The principle of this treatment is the increase in solubility of calcium phosphate brought about by the presence of higher concentrations than normal of urinary glucuronosides. The experimental biochemical work stated earlier in this report is sufficiently convincing to warrant further study and wider trial of this therapeutic principle. It would be rank presumption to state that we have demonstrated the efficacy of the regimen described above. The drug and the dosage were selected on a theoretical and tentative basis, and we admit some degree of surprise that this early series of patients had done so well. Our limited experience with salicylamide suggests that it may have some advantages over aspirin. With equivalent dosage, the output of total glucuronic acid in the urine is somewhat greater and the irritant effect upon the gastrointestinal tract is recognized to be less. The common stone-forming compounds in calcium-containing calculi are calcium oxalate, calcium phosphate and magnesium ammonium phosphate. We have not studied the solvent effect of glucuronosides on calcium oxalate and magnesium ammonium phosphate. We are certain that calcium phosphate was a component of the calculi in all but one patient in the series of 19. Chemical analysis, or preferably crystallographic analysis, of the calculus appears to us to be a necessary prerequisite, while the patient's co-operation in such matters as fluid intake and restriction of dietary calcium seems fully as important as the -regular use of the drug. A word of warning is necessary in the use of acetylsalicylic acid. Dosages in excess of 2 gm. may cause hypercalcinuria which is undesirable. We have occasionally noted a slight increase in urinary calcium excretion on thi.'l dosage. We believe that until we become more familiar with this type of treatnient, it seems quite necessary that the calcium and glucuronoside output in the tlrine be measured at reasonable intervals to determine the efficacy of the therapy. For glucuronoside determination we use and recommend the method of Fishman. 9 Urinary calcium was determined by the method of McCrudden.22 (The Sulkowitch test will be inadequate.) SUMMARY
A major objective .in the treatment of cases of recurrent calcium-containing calculi is the prevention of supersaturation of the urine with stone forming substances. Evidence from biochemical literature indicates that the solubility of calcium phosphate in the urine is increaset.i in the presence of glucuronosides. H Hawk, P. B., Oser, B. L., and Summe:•:son, W. H.: Practical Physiological Chemistry, 13th ed. New York and Toronto: Blakiston, 1954, p. 961.
446
EDWIN L. PRIEN AND BURNHAM S. WALKER
Of the numerous substances which augment the output of glucuronosides in the urine, derivatives of salicylic acid such as acetylsalicylic acid and salicylamide, appear to be highly effective and relatively nontoxic. A dosage of 2 gm. of acetylsalicylic acid in three divided doses, or an equivalent amount of salicylamide, is suggested, in addition to the usual dietary measures advised for such patients. Under such a regimen no growth or new formation of calculi occurred over a period of 12 to 18 months, in 17 out of a series of 19 patients with consistent histories of recurrent calcium-containing calculi.
Vol. 74, No. 4, October 1956 Printed in U.S.A.
STUDIES IN UROLITHIASIS: IV. URINARY GLUCURONOSIDES AND CALCIUM PHOSPHATE EDWIN L. PRIEN
AND
BURNHAM S. WALKER
From Boston University School of Medicine, Boston, Mass.
The actual cause of calculus formation in the urinary tract remains unknown in the great majority of cases. Hypersecretion of crystalloids of low solubility, or their decrease in solubility because of extremes of pH, are mechanisms and not causes. Under these circumstances, attempts to prevent recurrence of urinary calculus are based upon prevention of excessive supersaturation1 • 2 of the urine by substances which might precipitate to form stone. It is assumed, of course, that the well-recognized diagnostic and therapeutic measures have been carried out. These would include surgical removal of parathyroid adenomas and urinary tract obstructions, treatment of infections, and mobilization of recumbent patients. In a certain small number of cases a cure will be obtained by these therapies; there will be no more stone. However, in the majority of cases further treatment must consist of attempting to lower the urinary saturation of the offending crystalloids, by decreasing their output, increasing their solubility in the urine, or both. For calcium-containing stone, and this includes all crystalline calculi except uric acid and cystine, we may decrease the calcium or phosphorus in the diet or prevent the absorption of phosphorus by administration of the aluminum gels. We may also attempt to increase the solubility of calcium phosphate and magnesium ammonium phosphate by acidification therapy. The solubility of calcium oxalate is uninfluenced by changes in pH over the normal range. If a urea-splitting infection is present, the urine may not be rendered acid by any therapy other than eradication of the infection. For uric acid and cystine stone only alkalinization of the urine is of value. Hyaluronidase therapy 3 based on a theory of doubtful foundation 4 has also been tried with equivocal results. 6 In general, the prevention of recurrence of calcium-containing calculi by the various suggested regimens has not been highly successful; many physicians believe that such regimens are useless and prescribe only a large fluid intake. Our results have not been entirely unfavorable with some of these regimens, probably because of an intense interest in the subject. Their limitations have stimulated a search for some other way of decreasing The research reported in this paper was supported by a grant-in-aid from the American Urological Research Foundation, Inc. Read at annual meeting, American Urological Association, Los Angeles, Calif. May 19, 1955. 1 Meyer, J.: tl'ber die Ausfii.llung von Sedimenten und die Bildung von Konkrementen in den Harnwegen . Ztschr. f . klin. Med., 111: 613---687, 1929. 2 Prien, E. L. : Studies in urolithiasis: III. Physicochemical principles in stone formation and prevention . J. Urol., 73: 627-652, 1955. 3 Butt, A. J., Seifter, J . and Hauser, E . A.: Some recent advances in etiolo~y and treatment of kidney stones . Ohio State Med . J., pp . 21-24, 1955. 4 Smiddy, F. G.: Hyaluronidase and urinary stone. Proc. Roy. Soc. Med., 47: 807-809, 1954. 5 Prien, E. L . : Use of hyaluronidase to prevent urinary calculi. Report of a case in which stone recurrence was facilitated by hyaluronidase therapy. J .A.M .A., 154: 744-747, 1954. 440
STUDil:B IN UROLITHIABIS
441
the saturation of the urine of patients with recurrent calcium stone. We have reasoned that the ideal therapy would be one which did not interfere with normal homeostatic mechanisms by interfering with the absorption of calcium or phosphorus from the bowel, was not dependent on the maintenance of an acid urine, would be effective in an alkaline urine, did not require the daily ingestion of an unpleasant medication, could be taken orally in the form of tablets or capsules, was harmless in proper dosage, and not an excessive financial burden. GLUCURONOBIDEB
We learn from the work of others that increasing the concentration of glucuronosides in the urine should increase the solubility of calcium salts. Glucuronosides contain glucuronic acid, and are formed by the conjugation of this substance with any one of
1
HOH7 HCOH
I
HOCH
O Glucuronic acid
H6:JH H6
I
COOH many phenols and alcohols, of either metabolic or extraneous origin. Glucuronic acid conjugation is the most common of the chemical changes usually designated by the term "detoxication," in which a foreign compound in the body is changed to a derivative which is excreted in the urine. The glucuronic acid which is necessary for the biosynthesis of urinary glucuronosides is formed in the body from 3-carbon intermediates originating from glucose or glycerol. 8 An immediate metabolic precursor of urinary glucuronosides is uridine diphosphate glucuronic acid. This substance reacts with a suitable phenolic or alcoholic acceptor forming the glucuronoside with liberation of uridine and inorganic phosphate.7 Glucuronosides are normal urinary components. Free glucuronic acid is not found in the urine unless the substance itself or its lactone has been fed. Even under these circumstances about 80 per cent of a 5 gm. dose is not recovered in the urine. 8 Glucuronic acid combined as urinary glucuronosides may vary from 0.3 to 0.75 gm. per 24 hours, as reported for a single individual. 9 Somewhat larger ranges have been reported when groups have been studied. 10 8 Doerschuk, A. P.: Radiotracer studies of the biosynthesis of conjugated o-glucuronic acid. J. Biol. Chem., 195: 855-859, 1952. 7 Storey, I. D. E . and Dutton, G. J .: Uridine compounds in glucuronic acid metabolism. 2. The isolation and structure of 'uridine-diphosphate-glucuronic acid'. Biochem. J., 59:
279-288, 1955. 8 Ormsby, A. A.: The metabolism of glucuronic acid in man. Texas Rep. Biol. & Med., 10: 66-72, 1952. 9 Fishman, W. H ., Smith, M., Thompson, D ., Bonner, C. D., Kasdon, S. C. and Homburger, F.: Investigation of glucuronic acid metabolism in human subjects. J. Clinical Investig., 30: 685-696, 1951. 10 Artz, N- . E . and Osman, E. M.: Biochemistry of Glucuronic Acid. New York: Academic Press, Inc., 1950.
442
EDWIN L. PRIEN AND BURNHAM S. WALKER
After surveying the large number of compounds known to be excreted as urinary glucuronosides11 we decided that salicylate best met our specifications of easy availability and low toxicity. Salicylate is excreted in the urine with from 15 to 40 per cent of the total excretion conjugated with glucuronic acid. 12 Salicylate is also excreted partly as a glycine conjugate, salicyluric acid, partly as a metabolic product, gentisic acid, and partly as unchanged salicylate. 13 Acetylsalicylic acid yields similar excretory compounds. 14 Salicylamide is excreted as the glucuronoside, as the sulphate, and as the glucuronoside of its metabolic product gentisamide. 16 In our clinical studies to be summarized later, acetylsalicylic acid was used in the majority of cases to increase the urinary output of glucuronosides. In a few instances salicylamide was used. In neutral or alkaline aqueous media, calcium carbonate and calcium phosphate are insoluble. The presence of glucuronides in solution increases the solubility of these substances. 16 Complex glucuronosides have a greater solvent effect than simple glucuronic acid.17 The precipitation of calcium salts in urine as a result of bacterial decomposition of soluble complexes has been observed. 18 Urinary glucuronosides have been observed to be destroyed by Staphylococcus albus19 and many other bacterial species may be presumed to act in a similar manner. The maximum increase in solubility of calcium phosphate brought about by the presence of a glucurononoside has been reported at 43 per cent. 20 THERAPY
For the last 20 months we have treated patients with recurrent calcium-containing calculi by using a daily dosage of 2 gm. of acetylsalicylic acid divided into 3 doses a day. The doses were put in capsules, the aspirin having first been incorporated as a powder into a mixture of harmless excipients for the purpose of disguising it. All patients who received this medication were informed that it was experimental and harmless, but the name of the drug was not divulged. We wished to impress upon the patients the importance of regular use of the drug, and we feared that the drug was too familiar to be impressive. The patients were also instructed to drink fluids, other than milk, liberally. Moderation was urged 11 Teague, R. S.: The conjugates of o-Glucuronic acid of animal origin. Advances in Carbohydrate Chemistry, 9: 185-246, 1954. 12 Alpen, E. C., Mandel, H . G., Rodwell, V. W. and Smith, P. K.: The metabolism of C" carboxyl salicylic acid in the dog and in man. J. Pharmacol., 102: 150-155, 1951. 13 Kapp, E. M . and Coburn, A. F.: Urinary metabolites of sodium salicylate. J. Biol. Chem., 146: 549- 565, 1942. 14 Smith, M. J . H . : Some recent advances in the pharmacology of salicylates. J. Pharm. Pharmacol., 6: 81- 93, 1953. 16 Becher, A., Mikseh, J., Rambacher, P. and Schafer, A.: 'Ober das Verhalten des Salicylamid in Stoffwechsl des Menschen. Klin. Wochschr., 30: 913-918, 1952. 16 Mandi, I., Grauer, A. and Neuberg, C.: Solubilization of insoluble matter in nature. II. The part played by salts of organic and inorganic acids occurring in nature . Biochemica et Biophysica Acta, 10: 540-569, 1953. 17 Neuberg, C. and Grauer, A.: Enzymic cleavage of conjugated glucuronic acids in relation to the problems of mineralysis. Enzymologia, 16: 115-121, 1952. 18 Flocks, R.H.: Nature of urinary calcium: its role in calcium urolithiasis. J. Urol., 64: 633- 637, 1950. 19 Barber, M., Brooksbank, B. W. L . and Haslewood, G. A. D. : Destruction of urinary glucuronide by bacteria. Nature, 162: 701-702, 1948. 2 ° Cessi, C.: La solubilizzazione del fosfato tricalcico durante la sintesi di un glucuronide studiata con fosforo radioattivo. Boll. soc. ital. biol. sper., 30: 629-632, 1954.
STUDIES IN UROLITHIASIS
443
in use of milk or cream with coffee or cereal, and in the use of ice creams, dried fruits, nuts, chocolates, and sea foods. Butter was allowed freely but cheese was forbidden. This high fluid, low calcium diet, together with the aspirin capsules, constituted the entire therapeutic program for the majority of our patients. The few exceptions will be noted in the following section. RESULTS
The treatment just outlined has been prescribed for patients who have had frequently recurring calcium-containing calculi and for a small series of 6 patients in whom alkaline encrusting cystitis developed in the presence of inlying urethral catheters. The catheters became obstructed by calcium phosphate and magnesium ammonium phosphate deposits, requiring that the catheters be changed at intervals of 1 or 2 weeks. In all six of these patients there was satisfactory reduction of the encrustation.* None of these patients was uremic. We have made no trials of aspirin therapy in uremic patients and we do not recommend it since there is danger from failure of renal excretion of the drug. Our stone patients were ambulatory and were selected for this therapy on the basis of the history of recent recurrences of calcium-containing stone. In this report we will consider only 19 of these patients, this being the number who have received the therapy continuously for 1 year or more. We plan to report in the future on the larger series of patients when they have been under treatment long enough for the results to be reasonably conclusive. Of these 19 patients, all but two have a history of at least three separate recurrences. One of these patients had passed over a hundred stones in 14 years. Twelve of these patients had collectively required twenty-three major operations on kidneys or ureters for removal of calculi. Six of this group had presumably permanent scarring of the upper urinary tract, ureteral stricture, residual hydronephrosis, or gross deformity of the pelves or calyces. None of these patients had gone a year without a recurrence of a urinary calculus and some were carrying calculi at the time. All of these patients had pyelographic studies before the treatment was started, supplemented by measurements of serum calcium, serum inorganic phosphorus, and nonprotein nitrogen. Also, two 24-hour urine specimens were collected and studied on each patient while he was on the high-fluid, low-calcium diet, but before aspirin was given; these constituted the control or pre-treatment specimens. The measurements carried out included volume, pH, calcium, inorganic phosphate, magnesium, and total glucuronic acid. 9 This last mentioned analysis includes of course the normal urinary glucuronosides plus the extra glucuronosides produced as a result of the administration of salicylate, all expressed as glucuronic acid. These same analyses were repeated on 24-hour specimens collected after 2 weeks of treatment and at intervals thereafter, at least once in 3
* We have subsequently encountered several bedridden patients in whom encrustation of catheters was not prevented by salicylate therapy. It is probable that the glucuronide solubilizing mechanism may be inadequate to prevent precipitation of salts when present in excessive amounts in very alkaline urine. The remarkable effect of pH on solubility of the phosphate salts in urine is emphasized in the previous paper in this series (J. Urol., 73: 640, 1955); normal urine is 22 times supersaturated with calcium phosphate at pH 8.
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EDWIN L. PRIEN AND BURNHAM 8. WALKER
months. Some of the patients who lived at a distance were followed under the care of their local physicians who supervised the collection of 24-hour specimens, sterilized them, and sent us aliquot portions by mail. The output of total glucuronic acid ranged from 0.2 to 0.9 gm. per 24 hours in the patients who had not started aspirin therapy. While the patients were receiving aspirin the outputs ranged from 0.6 to 2.2 gm. per 24 hours. It was almost always possible to double the output and frequently to triple or quadruple it. Following a year or more of treatment all of these patients had plain urograms of the kidneys, ureters and bladder, excretory urograms, or both to determine if existing calculi had grown or new stones had appeared. These films were compared with those obtained before drug therapy was started. In 17 of these 19 patients, no new calculi appeared, nor was there increase in size or density of calculi present at the start. In one of the other two patients, there was a slight increase in size of his small renal calculi after 18 months under treatment. In this particular patient we were never able to achieve the usual increase in the output of glucuronic acid. We have no reason to think that this patient did not take the drug faithfully. It seems more reasonable to conclude that he is a poor glucuronoside producer or that there was excessive destruction of glucuronosides by bacterial action. The one remaining patient was a complete failure, although his glucuronoside production was excellent. His calculi became larger and additional calculi made their appearance. This patient differs from the others in the series in respect to the composition of his calculi. The other patients had produced stones composed chiefly of the usual type of calcium phosphate, or apatite stone. 21 We may call this basic calcium phosphate because it contains hydroxyl (OH) radicles. The patient in whom the therapy failed formed calculi composed of acid calcium phosphate (CaHP04). This compound occurs in less than 2 per cent of human renal calculi. Out of this group of 19 patients, 16 had infected urines when treatment was started. The organisms cultured included Pseudomonas aeruginosa, Proteus vulgaris, Micrococcus pyogenes, E. coli, Aerobacter aerogenes, Lactobacilli and diptheroids. Most of these infections had been of long standing and the patients had acquired strains resistant to the common antibacterial agents. One patient's urine was cleared of infection by supplemental antibacterial therapy during the period of treatment. Several of the other patients with infected urines became clear of the infection without additional therapy. In this group and in the larger group not presently being reported, there have been relatively few undesirable side effects of aspirin therapy. Signs of salicylate poisoning have been watched for, but for the most part have not appeared. Only one patient reported a transient ringing in the ears. Prothrombin time was determined and found normal in several patients with borderline renal function. No instance of granulocytopenia was noted. Several patients have reported 11 Prien, E. L. and Frondel, C.: Studies in urolithiasis. I. The composition of urinary calculi. J. Urol., 67: 949-991, 1947.
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gastric discomfort or heartburn, and two of these have been carried successfully on salicylamide, with the production of satisfactory outputs of urinary glucuronosides. Therapy had to be discontinued in one patient on account of an exacerbation of a duodenal ulcer which had previously caused no symptoms. DISCUSSION
This incomplete and preliminary report on a small group of patients is published at this time with the simple purpose of suggesting a new line of therapeutic approach to the problem of recurrent calcium-containing stones. The principle of this treatment is the increase in solubility of calcium phosphate brought about by the presence of higher concentrations than normal of urinary glucuronosides. The experimental biochemical work stated earlier in this report is sufficiently convincing to warrant further study and wider trial of this therapeutic principle. It would be rank presumption to state that we have demonstrated the efficacy of the regimen described above. The drug and the dosage were selected on a theoretical and tentative basis, and we admit some degree of surprise that this early series of patients had done so well. Our limited experience with salicylamide suggests that it may have some advantages over aspirin. With equivalent dosage, the output of total glucuronic acid in the urine is somewhat greater and the irritant effect upon the gastrointestinal tract is recognized to be less. The common stone-forming compounds in calcium-containing calculi are calcium oxalate, calcium phosphate and magnesium ammonium phosphate. We have not studied the solvent effect of glucuronosides on calcium oxalate and magnesium ammonium phosphate. We are certain that calcium phosphate was a component of the calculi in all but one patient in the series of 19. Chemical analysis, or preferably crystallographic analysis, of the calculus appears to us to be a necessary prerequisite, while the patient's co-operation in such matters as fluid intake and restriction of dietary calcium seems fully as important as the -regular use of the drug. A word of warning is necessary in the use of acetylsalicylic acid. Dosages in excess of 2 gm. may cause hypercalcinuria which is undesirable. We have occasionally noted a slight increase in urinary calcium excretion on thi.'l dosage. We believe that until we become more familiar with this type of treatnient, it seems quite necessary that the calcium and glucuronoside output in the tlrine be measured at reasonable intervals to determine the efficacy of the therapy. For glucuronoside determination we use and recommend the method of Fishman. 9 Urinary calcium was determined by the method of McCrudden.22 (The Sulkowitch test will be inadequate.) SUMMARY
A major objective .in the treatment of cases of recurrent calcium-containing calculi is the prevention of supersaturation of the urine with stone forming substances. Evidence from biochemical literature indicates that the solubility of calcium phosphate in the urine is increaset.i in the presence of glucuronosides. H Hawk, P. B., Oser, B. L., and Summe:•:son, W. H.: Practical Physiological Chemistry, 13th ed. New York and Toronto: Blakiston, 1954, p. 961.
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Of the numerous substances which augment the output of glucuronosides in the urine, derivatives of salicylic acid such as acetylsalicylic acid and salicylamide, appear to be highly effective and relatively nontoxic. A dosage of 2 gm. of acetylsalicylic acid in three divided doses, or an equivalent amount of salicylamide, is suggested, in addition to the usual dietary measures advised for such patients. Under such a regimen no growth or new formation of calculi occurred over a period of 12 to 18 months, in 17 out of a series of 19 patients with consistent histories of recurrent calcium-containing calculi.