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Hydroxyurea and infected stones
PRELIMINARY COMMUNICATION
HYDROXYUREA M. J. V. SMITH,
M.D.,
AND INFECTED
STONES
PH.D.
From the Division of Urology, Medical College of Virginia, Richmond, Virginia
ABSTRACT - The formation of struvite stones is closely linked to the enzyme m-ease produced by certain bacteria found in urine. It has been shown that hydroxamic acids wiZl block the activity of this enzyme. One of these, hydroxyurea, in a morning dose of 500 mg. was used in 21 patients for three months or longer. Most were then able to acidify their urine despite persistent urinary infection. Aft er three months, treatment with nitrofurantoin (Macrodantin and hydroxyurea (Hydrea), 78.6 per cent of patients had persistently sterile urine. There are 11 patients who have been observed for one year, and no toxic effect of the drug has been found. Three patients have shown some decrease in size of their retained calculus. This experimental study has shown that hydroxyurea may be useful in the management of struvite stones and deserves further study.
Every urologist is aware that after surgery for infected stones the recurrence rate of infection is extremely high. Struvite or magnesium ammonium phosphate stones are usually associated with urinary tract infections. The bacteria involved have the ability to produce an enzyme urease.’ This enzyme splits urea and produces an alkaline urine with high levels of ammonium ions. Elliot, Sharp, and Lewis2 have shown that urine becomes supersaturated above pH 7.1 and struvite will be formed. They also pointed out that struvite will dissolve when placed in urine below this pH. The class of compounds known as hydroxamic acids are specific and effective inhibitors of bacterial urease. 3 Griffith and his associates4+ have studied acetohydroxamic acid (AHA) and have shown in vitro and in vivo that this compound will inhibit urease and alter the pH of the urine. Their preliminary work in man and animals has shown that it is possible to acidify the urine and in a few instances dissolve a struvite calculus. Our initial studies confirmed their findings. Unfortunately acetohydroxamic acid is not readily available for full-scale studies. It, therefore, seemed a logical approach to use a similar hydroxamate, hydroxyurea (Hydrea). This drug has a well-characterized toxicity and is in general use. It is known that up to 80 per cent of hydroxyurea is excreted unchanged in the
274
urine within twenty-four hours.’ Also the toxic effect on the bone marrow elements is readily reversible. Preliminary Studies All patients who were taking hydroxyurea in the medical oncology clinic were screened, and it was found that they excreted acid urine (pH below 6), whether or not there was infection present in the urine. Five patients with proved proteus urinary infection were studied as follows. The pH of their urine was measured by means of nitrazine paper for two days. On the morning of the third day, a single oral dose of 500 mg. of hydroxyurea was administered. Their urinary pH was then monitored for the following seven days. The dose was increased at weekly intervals to 1.5 Gm. All were able to acidify their urine below pH 6 in spite of any untreated proteus infection. Interestingly this ability did not appear to be dose related. On the basis of these findings and because of two previous long-term studies, we were not anxious to exceed a daily dose of 1 Gm. of hydroxyurea.s,’ Clinical Studies All patients who entered the study had a renal calculus that was radiopaque and which UROLOGY/ MARCH1978 I VOLUMEXI, NUMBER
3
was presumed to be struvite. This presumption was based on the finding of residual stone after surgery or because of the characteristic roentgenographic appearance of a dendritic calculus associated with a urinary infection due to urease-producing bacteria. All patients had demonstrated consistently alkaline urine (pH greater than 7.0) in spite of all the usual efforts to obtain anacidic urine, for example, long-term antibiotic therapy or ascorbic acid supplements. They all had infected urine. Patients were excluded if (1) the creatinine clearance was below 50, (2) they were contemplating pregnancy or fatherhood, because of the possible teratogenic effect of the drug, or (3) the results of a metabolic evaluation were abnormal. They were then instructed to measure and record the pH of every voided urine by means of nitrazine paper. The experimental nature of the study was explained to all patients and informed consent obtained. The patients were given hydroxyurea, 500 mg. in the morning. No patient was given antibiotic therapy for the first three months of the study unless the clinical condition was such that it was essential. The patients measured and recorded the pH of each voided urine for twenty-four hours once a week. They were seen at monthly intervals, and at this time the urine was cultured and a complete blood count and an automated serum biochemical profile obtained (blood sugar, creatinine, blood urea nitrogen, sodium, potassium, carbon dioxide, chloride, uric acid, calcium, phosphorus, total protein, albumin, cholesterol, total bilirubin, alkaline phosphatase, serum glutamic oxaloacetic transaminase, and lactic dehydrogenase). A plain film of the abdomen was obtained at three monthly intervals. At the same time a twenty-four-hour urine collection for calcium, phosphorus, magnesium, uric acid, oxalate, and creatinine was studied. After three months, the patient was begun on methenamine hippurate (Hiprex) or nitrofurantoin (Macrodantin). Results Twenty-one patients have been entered in the protocol for three months or longer, and the findings form the basis of this report. There were 6 patients who were unable to persist in the study and are reported as controls. That is to say they gave up hydroxyurea but con-
UROLOGY
/ MARCH 1978 / VOLUME
XI. NUMBER 3
tinued with antibiotics. Two patients were lost to follow-up. All patients reported that their urine could be maintained in the range of pH 5.0 to 6.5 on all voided specimens. A pH 6.5 was usually found in the early morning specimen. However, it was not true that this was a consistent finding while patients were taking hydroxyurea alone. Nine patients always had an acid urine (pH below 6.0) throughout the study, 6 had acid urine on all specimens except occasionally in the morning, while the remainder would have breakthrough episodes of alkaline urine (pH above 7). In 1 patient a transiently sterile urine developed with hydroxyurea alone. All the others continued to grow bacteria on urine culture. Almost uniformly the number of white cells seen in random urine samples has fallen in spite of persistent infection. Patients have reported an improved sense of well being while on the drug, but there was no objective evidence to support these statements and this finding probably represents a placebo effect. Initially, we added methenamine hippurate at three months, and in 4 of 11 patients sterile acid urine developed. After these results were reviewed we elected to change to nitrofurantoin. Three months after the use of hydroxyurea and nitrofurantoin, 11 of 14 patients had sterile urine. There are 11 patients who have been on the protocol a year or longer, and in three there has been measurable decrease in the size of the calculus. This change has not been seen before six months. In 2 patients there has been an increase in size of the calculus while on hydroxyurea alone. One of these has had the calculus removed by surgery. Crystallographic studies revealed the removed stone had a central portion of struvite which was covered by apatite. There have been no long-term toxic effects in any of the patients studied. There has been no evidence on serial blood studies of marrow depression or altered liver or renal function as a result of taking 500 mg. of hydroxyurea daily. Three patients were unable to continue the drug because of persistent morning nausea, and in 1 patient headaches and myalgia developed. In all 4 patients their symptoms developed within one week of starting therapy. They are considered failures but have been followed according to our protocol and act as prospective controls.
275
Comment Fishbein and Carbone had shown that hydroxyurea was both an inhibitor and a possible substrate for bacterial urease when studied in vitro. These studies were, therefore, approached with considerable caution and were conceived as a pilot trial, particularly since it is theoretically possible for hydroxyurea, which is known to be labile and might undergo spontaneous or enzymatic hydrolysis, to form free hydroxylamine and ammonium ions. Other investigators have pointed out that up to 80 per cent of the therapeutic dose could be recovered from the urine in twenty-four hours.’ Fishbein and Carbone’O had pointed out earlier that hydroxyurea could be hydrolyzed to hydroxylamine which then would react with thioesters to give acetohydroxamic acid. We have also detected acetohydroxamic acid in the urine of our patients, but not on a consistent basis. This finding is currently under further investigation because of its implications. What compound is actually providing the anti-urease activity seen in our group of patients? It may well be that the apparent variations in urine pH and anti-stone activity that are reported in this study are due to variations in the levels of AHA and/or hydroxylamine found in the urine, not hydroxyurea. Interestingly there was a decrease in the pyuria seen in these patients. This finding would seem to lend support to the work of Maclaren” and Griffith and Musher.5 They have pointed out that urease plays a role in the pathogenesis of pyelonephritis. They have shown in rats that pyelonephritis due to a nonurease-producing proteus strain will induce minimal changes in the renal parenchyma. The significant ease with which the urine was rendered sterile by nitrofurantoin is important. In none of these patients prior to entry in the study nor in our controls have we been able to keep the urine consistently sterile. This supports the earlier reports that hydroxamic acid will potentiate the bacteriostatic activities of simple antibiotics. 4~11 So far, we have not chosen to take these patients off nitrofurantoin after the urine becomes sterile, because the basic effort was to see whether or not struvite will dissolve and thus develop an oral therapeutic attack on recurrent or remaining infected stones. We currently have a protocol underway to see whether or not the use of hydroxamic acid
276
alone is the more desirable way to manage an infected stone after initial sterilization of the urine. Theoretically, these substances should be present in the urine in sufficient concentration to inhibit or to prevent any reinfection from the “trapped’ bacteria found within the interstices of the stone. Nemoy and Stamey12 have presented convincing evidence that these bacteria retain their pathogenicity; therefore, as the stone is dissolved, they may become a problem, however their urease activity will be inhibited. A further question which this study has not answered is related to matrix of the stone. As the struvite crystals are resorbed the inherent matrix of the calculus will be uncovered. In theory, this matrix could then provide a nidus for the formation of a calculus with a different crystalline makeup. This may explain the apparent stone growth that occurred. Both patients had urinary calciums that were above 150 mg. in twenty-four hours as opposed to the other patients whose urinary calciums were below this normal level. It is possible that in these patients the ambient pH was optimum for the deposition of apatite. This complication is of such import that no patient with a urinary calcium above 150 mg. will be treated until this complication has been better explained. These preliminary findings and earlier work3,10 raise the question as to whether or not these effects could be better obtained by using a lower dosage of hydroxyurea (for example, 250 mg. twice a day) spread throughout the day. Unfortunately, it is not produced in a low-dosage form at this time. In conclusion, it must be stressed that this study is strictly experimental in nature. Hydroxyurea will in selected patients reverse the alkalinity of the urine. It appears to act in a synergistic way with an appropriate antibiotic to control urinary infection. Whether or not the promise of being able to dissolve existing stones will be substantiated remains to be seen. Hydroxyurea is a potentially dangerous drug, and its long-term use in low dosage for periods up to one year has been without toxic effects. It is obvious that a longer period of study is mandatory before such an approach can be recommended for universal use. Richmond, Virginia 23298 ACKNOWLEDGMENT. My gratitude to Dr. Floyd Fried, of Chapel Hill, for planting the seed from which these ideas grew.
UROLOGY
I
MARCH 1978 i
VOLUME
XI, NUMBER 3
References 1. Chute R, and Suby HI: Prevalence and importance of ureasplitting bacterial infections of the urinary tract in the formation of calculi, J. Ural. 44: 590 (1940). 2. Elliot JS, Sharp RF, and Lewis L: The solubility of struvite in urine, ibid. 81: 366 (1959). 3. Fishbein WN, and Carbone PP: Urease catalysis. II. Inhibition of the enzyme by hydroxyurea, hydroxylamine, and acetohydroxamic acid, J. Biol. Chem. 240: 2407 (1965). 4. Griffith DP, Musher DM, and Campbell JW: Urease, the primary cause of infection-induced stones, Invest. Urol. 13: 346 (1976). 5. Griffith DP, and Musher DM: Acetohydroxamic acid: potential use in urinary infection caused by urea-splitting bacteria, Urology 5: 299 (1975). 6. Griffith DP, Gibson JR, Clinton CW, and Musher DM:
UROLOGY
/
MARCH
1978
/
VOLUME
XI, NUMBER
3
Acetohydroxamic acid: initial investigations in man, in Fleisch, H, et al., Eds.: Urolithiasis Research, New York, Plenum Press, 1976. 7. Rosner F, Rubin H, and Parise F: Studies on the absorption, distribution, and excretion of hydroxyurea, Cancer Chemother. Rep. 55: 167 (1971). 8. Dahl MGC, and Comaish JS: Long-term effects of hydroxyurea in psoriasis, Br. Med. J. 4: 585 (1972). 9. Moschella SL, and Greenwald MA: Psoriasis with hydroxyurea, Arch. Dermatol. 107: 363 (1973). 10. Fishbein WN, and Carbone PP: Hydroxyurea, mechanism of action, Science 142: 1069 (1963). 11. MacLaren DM: The influence of acetohydroxamic acid on experimental pyelonephritis, Invest. Urol. 12: 146 (1974). 12. Nemoy NJ, and Stamey TA: Surgical, bacteriological and biochemical management of infection stones, J.A.M.A. 215: 1470 (1971).
277
HYDROXYUREA M. J. V. SMITH,
M.D.,
AND INFECTED
STONES
PH.D.
From the Division of Urology, Medical College of Virginia, Richmond, Virginia
ABSTRACT - The formation of struvite stones is closely linked to the enzyme m-ease produced by certain bacteria found in urine. It has been shown that hydroxamic acids wiZl block the activity of this enzyme. One of these, hydroxyurea, in a morning dose of 500 mg. was used in 21 patients for three months or longer. Most were then able to acidify their urine despite persistent urinary infection. Aft er three months, treatment with nitrofurantoin (Macrodantin and hydroxyurea (Hydrea), 78.6 per cent of patients had persistently sterile urine. There are 11 patients who have been observed for one year, and no toxic effect of the drug has been found. Three patients have shown some decrease in size of their retained calculus. This experimental study has shown that hydroxyurea may be useful in the management of struvite stones and deserves further study.
Every urologist is aware that after surgery for infected stones the recurrence rate of infection is extremely high. Struvite or magnesium ammonium phosphate stones are usually associated with urinary tract infections. The bacteria involved have the ability to produce an enzyme urease.’ This enzyme splits urea and produces an alkaline urine with high levels of ammonium ions. Elliot, Sharp, and Lewis2 have shown that urine becomes supersaturated above pH 7.1 and struvite will be formed. They also pointed out that struvite will dissolve when placed in urine below this pH. The class of compounds known as hydroxamic acids are specific and effective inhibitors of bacterial urease. 3 Griffith and his associates4+ have studied acetohydroxamic acid (AHA) and have shown in vitro and in vivo that this compound will inhibit urease and alter the pH of the urine. Their preliminary work in man and animals has shown that it is possible to acidify the urine and in a few instances dissolve a struvite calculus. Our initial studies confirmed their findings. Unfortunately acetohydroxamic acid is not readily available for full-scale studies. It, therefore, seemed a logical approach to use a similar hydroxamate, hydroxyurea (Hydrea). This drug has a well-characterized toxicity and is in general use. It is known that up to 80 per cent of hydroxyurea is excreted unchanged in the
274
urine within twenty-four hours.’ Also the toxic effect on the bone marrow elements is readily reversible. Preliminary Studies All patients who were taking hydroxyurea in the medical oncology clinic were screened, and it was found that they excreted acid urine (pH below 6), whether or not there was infection present in the urine. Five patients with proved proteus urinary infection were studied as follows. The pH of their urine was measured by means of nitrazine paper for two days. On the morning of the third day, a single oral dose of 500 mg. of hydroxyurea was administered. Their urinary pH was then monitored for the following seven days. The dose was increased at weekly intervals to 1.5 Gm. All were able to acidify their urine below pH 6 in spite of any untreated proteus infection. Interestingly this ability did not appear to be dose related. On the basis of these findings and because of two previous long-term studies, we were not anxious to exceed a daily dose of 1 Gm. of hydroxyurea.s,’ Clinical Studies All patients who entered the study had a renal calculus that was radiopaque and which UROLOGY/ MARCH1978 I VOLUMEXI, NUMBER
3
was presumed to be struvite. This presumption was based on the finding of residual stone after surgery or because of the characteristic roentgenographic appearance of a dendritic calculus associated with a urinary infection due to urease-producing bacteria. All patients had demonstrated consistently alkaline urine (pH greater than 7.0) in spite of all the usual efforts to obtain anacidic urine, for example, long-term antibiotic therapy or ascorbic acid supplements. They all had infected urine. Patients were excluded if (1) the creatinine clearance was below 50, (2) they were contemplating pregnancy or fatherhood, because of the possible teratogenic effect of the drug, or (3) the results of a metabolic evaluation were abnormal. They were then instructed to measure and record the pH of every voided urine by means of nitrazine paper. The experimental nature of the study was explained to all patients and informed consent obtained. The patients were given hydroxyurea, 500 mg. in the morning. No patient was given antibiotic therapy for the first three months of the study unless the clinical condition was such that it was essential. The patients measured and recorded the pH of each voided urine for twenty-four hours once a week. They were seen at monthly intervals, and at this time the urine was cultured and a complete blood count and an automated serum biochemical profile obtained (blood sugar, creatinine, blood urea nitrogen, sodium, potassium, carbon dioxide, chloride, uric acid, calcium, phosphorus, total protein, albumin, cholesterol, total bilirubin, alkaline phosphatase, serum glutamic oxaloacetic transaminase, and lactic dehydrogenase). A plain film of the abdomen was obtained at three monthly intervals. At the same time a twenty-four-hour urine collection for calcium, phosphorus, magnesium, uric acid, oxalate, and creatinine was studied. After three months, the patient was begun on methenamine hippurate (Hiprex) or nitrofurantoin (Macrodantin). Results Twenty-one patients have been entered in the protocol for three months or longer, and the findings form the basis of this report. There were 6 patients who were unable to persist in the study and are reported as controls. That is to say they gave up hydroxyurea but con-
UROLOGY
/ MARCH 1978 / VOLUME
XI. NUMBER 3
tinued with antibiotics. Two patients were lost to follow-up. All patients reported that their urine could be maintained in the range of pH 5.0 to 6.5 on all voided specimens. A pH 6.5 was usually found in the early morning specimen. However, it was not true that this was a consistent finding while patients were taking hydroxyurea alone. Nine patients always had an acid urine (pH below 6.0) throughout the study, 6 had acid urine on all specimens except occasionally in the morning, while the remainder would have breakthrough episodes of alkaline urine (pH above 7). In 1 patient a transiently sterile urine developed with hydroxyurea alone. All the others continued to grow bacteria on urine culture. Almost uniformly the number of white cells seen in random urine samples has fallen in spite of persistent infection. Patients have reported an improved sense of well being while on the drug, but there was no objective evidence to support these statements and this finding probably represents a placebo effect. Initially, we added methenamine hippurate at three months, and in 4 of 11 patients sterile acid urine developed. After these results were reviewed we elected to change to nitrofurantoin. Three months after the use of hydroxyurea and nitrofurantoin, 11 of 14 patients had sterile urine. There are 11 patients who have been on the protocol a year or longer, and in three there has been measurable decrease in the size of the calculus. This change has not been seen before six months. In 2 patients there has been an increase in size of the calculus while on hydroxyurea alone. One of these has had the calculus removed by surgery. Crystallographic studies revealed the removed stone had a central portion of struvite which was covered by apatite. There have been no long-term toxic effects in any of the patients studied. There has been no evidence on serial blood studies of marrow depression or altered liver or renal function as a result of taking 500 mg. of hydroxyurea daily. Three patients were unable to continue the drug because of persistent morning nausea, and in 1 patient headaches and myalgia developed. In all 4 patients their symptoms developed within one week of starting therapy. They are considered failures but have been followed according to our protocol and act as prospective controls.
275
Comment Fishbein and Carbone had shown that hydroxyurea was both an inhibitor and a possible substrate for bacterial urease when studied in vitro. These studies were, therefore, approached with considerable caution and were conceived as a pilot trial, particularly since it is theoretically possible for hydroxyurea, which is known to be labile and might undergo spontaneous or enzymatic hydrolysis, to form free hydroxylamine and ammonium ions. Other investigators have pointed out that up to 80 per cent of the therapeutic dose could be recovered from the urine in twenty-four hours.’ Fishbein and Carbone’O had pointed out earlier that hydroxyurea could be hydrolyzed to hydroxylamine which then would react with thioesters to give acetohydroxamic acid. We have also detected acetohydroxamic acid in the urine of our patients, but not on a consistent basis. This finding is currently under further investigation because of its implications. What compound is actually providing the anti-urease activity seen in our group of patients? It may well be that the apparent variations in urine pH and anti-stone activity that are reported in this study are due to variations in the levels of AHA and/or hydroxylamine found in the urine, not hydroxyurea. Interestingly there was a decrease in the pyuria seen in these patients. This finding would seem to lend support to the work of Maclaren” and Griffith and Musher.5 They have pointed out that urease plays a role in the pathogenesis of pyelonephritis. They have shown in rats that pyelonephritis due to a nonurease-producing proteus strain will induce minimal changes in the renal parenchyma. The significant ease with which the urine was rendered sterile by nitrofurantoin is important. In none of these patients prior to entry in the study nor in our controls have we been able to keep the urine consistently sterile. This supports the earlier reports that hydroxamic acid will potentiate the bacteriostatic activities of simple antibiotics. 4~11 So far, we have not chosen to take these patients off nitrofurantoin after the urine becomes sterile, because the basic effort was to see whether or not struvite will dissolve and thus develop an oral therapeutic attack on recurrent or remaining infected stones. We currently have a protocol underway to see whether or not the use of hydroxamic acid
276
alone is the more desirable way to manage an infected stone after initial sterilization of the urine. Theoretically, these substances should be present in the urine in sufficient concentration to inhibit or to prevent any reinfection from the “trapped’ bacteria found within the interstices of the stone. Nemoy and Stamey12 have presented convincing evidence that these bacteria retain their pathogenicity; therefore, as the stone is dissolved, they may become a problem, however their urease activity will be inhibited. A further question which this study has not answered is related to matrix of the stone. As the struvite crystals are resorbed the inherent matrix of the calculus will be uncovered. In theory, this matrix could then provide a nidus for the formation of a calculus with a different crystalline makeup. This may explain the apparent stone growth that occurred. Both patients had urinary calciums that were above 150 mg. in twenty-four hours as opposed to the other patients whose urinary calciums were below this normal level. It is possible that in these patients the ambient pH was optimum for the deposition of apatite. This complication is of such import that no patient with a urinary calcium above 150 mg. will be treated until this complication has been better explained. These preliminary findings and earlier work3,10 raise the question as to whether or not these effects could be better obtained by using a lower dosage of hydroxyurea (for example, 250 mg. twice a day) spread throughout the day. Unfortunately, it is not produced in a low-dosage form at this time. In conclusion, it must be stressed that this study is strictly experimental in nature. Hydroxyurea will in selected patients reverse the alkalinity of the urine. It appears to act in a synergistic way with an appropriate antibiotic to control urinary infection. Whether or not the promise of being able to dissolve existing stones will be substantiated remains to be seen. Hydroxyurea is a potentially dangerous drug, and its long-term use in low dosage for periods up to one year has been without toxic effects. It is obvious that a longer period of study is mandatory before such an approach can be recommended for universal use. Richmond, Virginia 23298 ACKNOWLEDGMENT. My gratitude to Dr. Floyd Fried, of Chapel Hill, for planting the seed from which these ideas grew.
UROLOGY
I
MARCH 1978 i
VOLUME
XI, NUMBER 3
References 1. Chute R, and Suby HI: Prevalence and importance of ureasplitting bacterial infections of the urinary tract in the formation of calculi, J. Ural. 44: 590 (1940). 2. Elliot JS, Sharp RF, and Lewis L: The solubility of struvite in urine, ibid. 81: 366 (1959). 3. Fishbein WN, and Carbone PP: Urease catalysis. II. Inhibition of the enzyme by hydroxyurea, hydroxylamine, and acetohydroxamic acid, J. Biol. Chem. 240: 2407 (1965). 4. Griffith DP, Musher DM, and Campbell JW: Urease, the primary cause of infection-induced stones, Invest. Urol. 13: 346 (1976). 5. Griffith DP, and Musher DM: Acetohydroxamic acid: potential use in urinary infection caused by urea-splitting bacteria, Urology 5: 299 (1975). 6. Griffith DP, Gibson JR, Clinton CW, and Musher DM:
UROLOGY
/
MARCH
1978
/
VOLUME
XI, NUMBER
3
Acetohydroxamic acid: initial investigations in man, in Fleisch, H, et al., Eds.: Urolithiasis Research, New York, Plenum Press, 1976. 7. Rosner F, Rubin H, and Parise F: Studies on the absorption, distribution, and excretion of hydroxyurea, Cancer Chemother. Rep. 55: 167 (1971). 8. Dahl MGC, and Comaish JS: Long-term effects of hydroxyurea in psoriasis, Br. Med. J. 4: 585 (1972). 9. Moschella SL, and Greenwald MA: Psoriasis with hydroxyurea, Arch. Dermatol. 107: 363 (1973). 10. Fishbein WN, and Carbone PP: Hydroxyurea, mechanism of action, Science 142: 1069 (1963). 11. MacLaren DM: The influence of acetohydroxamic acid on experimental pyelonephritis, Invest. Urol. 12: 146 (1974). 12. Nemoy NJ, and Stamey TA: Surgical, bacteriological and biochemical management of infection stones, J.A.M.A. 215: 1470 (1971).
277