Experimental Study of Solvent Action of Versene on Urinary Calculi

THE JOURNAL OF UROLOGY

Vol. 65, No. 2, February, 1951 Printed in U.S.A.

EXPERIMENTAL STUDY OF SOLVENT ACTION OF VERSENE ON URINARY CALCULI BENJAMIN S. ABESHOUSE

AND

TOBIAS WEINBERG

From the Departments of Urology and Pathology, Sinai Hospital, Baltimore, Md.

The surgical treatment of urinary calculi is one of the most serious and perplexing problems in the entire realm of medicine. Medical history is replete with numerous articles on the prevention of the formation of urinary calculi and the non-surgical treatment of the condition. The various methods and procedures designed to effect dissolution of urinary calculi "in vivo" have in general failed in this purpose. In a few instances, dissolution or disintegration of urinary calculi have been accomplished by various methods. 1 Keyser has made an extensive study of the dissolution and disintegration of urinary calculi in vitro by irrigation with various inorganic acids, solvents or reagents with varying degrees of success. He also studied the effect of some organic acids and solutions upon the dissolution of urinary calculi (table 1). His observations revealed that most of the inorganic and organic acid or reagents were effective in producing prompt surface dissolution of most alkaline stones and that at this stage the calculus was found to be covered by a mucinous organic substance which interfered with further action of the solvent. He used various ferments, i.e., urease, pepsin, diastase, trypsin and papaya to dissolve or destroy this gelatinous organic envelop and to enhance further action of the inorganic and organic solvents. Urease was found to be the most effective enzyme in dissolution of urinary calculi in vitro and in vivo. It soon became apparent to the authors that the ideal solvent for urinary calculi in vitro and in vivo would be an agent or solution of either an inorganic or organic chemical nature which would possess the properties of dissolving most inorganic constituents (and perhaps some of the organic constituents) of urinary calculi and at the same time have the power of hydrolyzing or dissolving proteins, alkaline metal proteinates, etc. The search for such an agent invoked a study of the various chemicals employed commercially for the purposes of sequestration, chelation, dispersion, etc. After considerable study it was decided to test the efficacy of versene in the dissolution of urinary calcuil. This compound Read at annual meeting, American Urological Association, Washington, D. C., June 1, 1950. 1 1) Crowell (1924): Dissolution of cystine calculi by alkalinization of the urine and irrigation of the kidney with alkalies. 2) Randall (1932): Treatment of alkaline encrusted cystitis and dissolution of small bladder calculi by irrigations of 1 per cent phosphoric acid. 3) Keyser (1932): Dissolution and disintegration of a renal calcium phosphate calculus by irrigation of the kidney with dilute aqua regia and by acidifying the patient's urine with acid diet and dilute aqua regia by mouth. 4) Higgins (1935): Dissolution of alkaline earth stones by acid ash diet, acidifying drugs and large doses of vitamins A and D. A few acid stones were dissolved by an alkaline ash diet and alkalinizing drugs. 5) Albright, Suby and Sulkowitch (1939): Dissolution of complex alkaline earth phosphate and carbonate calculi by irrigation of the kidney with citric acid mixtures, i.e., citrate buffer solutions (Albright), solutions G and M (Suby). 6) Keyser (1942): Retrograde dissolution of calculi with ferments (enzymes), and citrate solution G. 316

3]7

SOLVENT ACTION OF VERSENE ON URINARY CALCULI

appeared to possess the highest efficiency as a solvent of the usual inorganic salts found in urinary calculi, and was a hydrolyzing agent of various protein compounds and thus offered the greatest promise as a solvent of urinary calculi. The present study represents the first effort to test the efficacy of any organic sequestering and chelating compound in the dissolution of urinary calculi. In the field of industrial chemistry, special attention has recently been focussed on the subject of sequestering and chelating agents particularly as applied to "softening" of water and correction of "boiler water imperfection." The difference between complexing, sequestration and chelating is essentially a matter of chemical structure. A complex is any material which will inactivate a metallic ion. The most common complexing agents are ammonia, citrates and tartrates. All sequestering and chelating agents are complexing agents but all complexing agents may or may not be sequestering or chelating agents. TABLE

1. Stndy of efj'ecl of vari01is solvents on iirinary calculi (6 hour treatrnenl) by Scherer, Cla:fj'ey and Keyser) SOL\'E:S-T

a,. b. c. d. e. f. g. h.

Citrate buffer (Albright). G solution (Suby) 1% citrate solution. Phosphate buffer. 1% tartaric acid. 5% tartaric acid. 1 % meta phosphoric acid .. Benzoic acid (saturated). 1 1% disodium phosphate j. 1% dihydrogen potassium phosphate.

%

DISSOLUTION

12.50 12.60 0.54

ORDER OF EFFICIENCY

2 1 9

LOO

8

3 .15 1.12 0.09 8.50 6.60 1.40

5 7 10

.,"

4

6

Sequestration is the ability to form soluble non-ionic complexes which are undissociated and do not posse,;s a heterocyclic ring strnctme. According to Daugherty, sequestration is the reduction in concentration of a multivalent positive ion in solution by combination ffith a negative ion to form a complex ion to the extent that the remaining concentration of multivalent prn,ihve ions is insufficient to be precipitated by a given negative ion with which it has a low solubility production, The multivalent positive ion usually disappears from solution by removal as a precipitate, but may also be removed by deposition as an element or evolution of a gas. Thus a sequestering agent is any compound which will inactivate a metallic ion and at the same time keep it in solution. Ammonia, cyanide, versene, triglycine and hexametaphosphate are sequestering agents. In Webster's Chemical Dictionary, chelation is defined as the chemical process by means of which a group or compound possessing two valences, principal or residual or both, attaches itself to a central metallic atom so as to form a heterocyclic ring structure "Within the molecule. Thus a chelating agent is a compound which has the ability to form soluble non-ionic compounds with polyvalent metal ions whose properties are completely changed in solution. The chelated metallic ions can no longer be precipitated from solution by the usual

318

BENJAMIN S. ABESHOUSE AND TOBIAS WEINBERG

precipitating agents. Dimethyl glyoxine, cupferron, triglycine and versene are examples of chelating agents. In most cases, the chelating compounds are very insoluble and their primary use in the past has been as organic precipitating agents. The presence of a heterocylic ring structure in the chelating agent serves to distinguish the process of chelation from sequestration. As early as 1833, Thomas Graham, the British chemist and founder of colloid chemistry, discovered a new complex form of phosphate, i.e., sodium metaphosphate, which is a glossy or amorphous compound having the empirical formula-N aPo3-with a molecular ratio of 1 Na20: 1 P 20&. In 1849 Fleitman considered the compound to be a hexametaphosphate. In 1929 Hall and Jackson first used this chemical commercially for the treatment of impurities and precipitates in boiler water and in 1932 Hall employed the compound to soften water without formation of precipitates and without removal of the "hardness" constituents. Other inorganic and organic sequestering agents containing phosphorus have been described; viz., (a) crystalline sodium pyrophosphate Na4P201 or Na2H2P201, (b) crystalline sodium tripolyphosphate Na5P 30 10 and (c) sodium phytate C6Hs(Na 3P04)s-inositol hexaphosphate. In 1938 Enders prepared and investigated the properties of two new sequestering and chelating agents which were organic compounds devoid of phosphorus. The first was designated as Trilon A, i.e.-N (Ch2CooNa) 3 and the second, Trilon B, which is also known as the sodium salt of ethylene diamine tetra acetic acid or sodium salt of ethylene bisimino-diacetic acid. The latter substance is better known in this country as versene which is the registered trademark of the Bersworth Chemical Co., Framingham, Mass., who are manufacturers of this synthetic polyaminoacid and its sodium salts. Although this substance has attained widespread industrial use, it has received very little attention in chemical and physiological textbooks and reviews. The various complex salts of this chelating agent were prepared by Pfeiffer and his coworkers in 1942, and Brintzinger et al. in 1942 and 1943. The acid-base properties and the equilibrium constants with hydrogen ion and with various metal ions have been investigated by Schwarzenbach and his coworkers in 1947 and 1948. CHEMISTRY OF VERSENE

Information concerning the chemical formula, reactions and properties of ethylene diamine tetra acetic acid and its sodium salts has been supplied by A. E. Martell, R. C. Plumb and F. C. Bersworth of the Bersworth Chemical Co., Framingham, Mass., and by J. J. Singer, chief chemist of this company. Versene is the aqueous solution of the tetra sodium salt of ethylene diamine tetra acetic acid. The chemical structure of this compound is usually represented as

SOLVm•,T ACTION OF VERSENE ON URI"ARY CALCULI

319

Since this is a salt of a strong base, it is completely ionized in solution. Consequently its structure may also be represented by

l

·-ooCCH2/

/

Ch2C00-1

N-Ch2-Ch2-N

~-OOCCH2/

/Ch2C00-_4Na+

For practical purposes, this formula may be contracted to ~ a 4Ve. H+ H+ H+ Na4Ve .---+ Ka,HVe .---+ Na2H2Ve :;=.. ---+ Hi,Ve OHOHOHThe sodium salt may be easily crystallized from water solution as a hydrate. Its structure is represented by NaOOCCH2

CH2COONa

/ HOOCCH2

N -CH2-CH2-N

/

/

/

CH2COOH

It has been definitely shown that the sodium in the bisodium salt is completely ionized in solution. It is also known that the two basic nitrogen atoms are in a position which is alpha to the carboxyl groups and that such groups exist in the form of "zwitter ions". Consequently the structure for this disodium salt may be written as OOCCH2/ /H

H/ /CH2C00-,2Xa+

N-CH2-CH2-N

/+ OOCCH2

+/ CH2COO-_

The sodium and other alkali metal salts of ethylene diamine tetra acetic acid are quite soluble in water. The acid itself is very insoluble. Thus the addition of any strong acid to a solution of a salt of versene results in the formation of a crystalline precipitate of the acid. For convenience the various salts and the acid may be called tetra sodium versene, disodium versene, versene acid, etc. COMPLEX FORIVIATIOK OF l\UJTAL IONS ,HTH VERSENE

'\Vhen a divalent metal ion, i.e., calcium ion, forms a complex with versene salts, the metal becomes coordinated with versene anion as indicated in following equation: Ca+++ Xa 4Ve---+ Na2 [CaVe] + 2 Ka+ In this manner, the calcium salt is bound up in a non-ionic complex with versene. A considerable drop in pH occurs when a neutral calcium salt, i.e., calcium chloride is added to a solution of disodium salt of versene. This is due to the liberation of hydrogen ions in the course of complex formation: Ca+++ Xa2H2Ve---+ Na2 [CaVe] + 2 H+ Thus the solution undergoes a considerable drop in pH and an equilibrium is reached.

320

BENJAMIN S. ABESHOUSE AND TOBIAS WEINBERG

It is important to bear in mind that the formation of acid in the course of the complex formation of metal salts with versene predicts the pH stability of versene complexes. The addition of acid to a solution of versene complex would obviously reverse the equilibrium indicated in the above equation. Hence it is readily apparent that all versene complexes become less stable as the pH is reduced and show maximum stability at high pH. This fact has an important bearing on the various applications for versene. It is important to note that most versene complexes are produced in solution in the form of their alkali-metal salts and hence are quite soluble. The metal ion undergoing complex formations is also tied up in a non-ionic form and hence will not precipitate even in the presence of negative ions which usually precipitate such a metal. It is also known that alkaline versene solutions will dissolve precipitates of many metals even though such precipitates usually have a maximum insolubility in bases. This is indicated in the following equations: Calcium phosphate Ca3(Po4)2 + 3Na4Ve - 3Na2 (CaVe)+ 2Na3Po4 Calcium oxalate CaC204 + 3Na4Ve - Na2 (CaVe)+ Ka2C204 Magnesium carbonate MgCo3 + No4Ve - Na2 (MgVe) + Ka2 Co3 The chelating or sequestering power of versene ·with ions of heavy metals, i.e., copper, nickel, cobalt, etc. is in general much stronger than with the alkaline earth metals listed above. It is also known that most metals are strongly sequestered above a pH of 5, while the heavy metals may be sequestered even at somewhat lower pH values. The higher the pH, the greater is the chelating power of versene with a particular metal. APPLICATION

The uses of sequestering agents in general have been described by Daugherty in 1948. Although versene and its alkali metal salts have most of the properties of ordinary sequestering agents, it differs in many respects and is more properly called a chelating agent. It is interesting to compare versene, a complex unnatural synthetic polyaminoacid with hexametaphosphate, a common type of inorganic sequestering agent. Martell, Plumb and Bersworth pointed out that the sequestering action of hexametaphosphate is due to some extent to the dispersion action of the reagents. Thus the precipitation of alkaline earth ions is prevented because the dispersing action of the finely divided precipitate tends to prevent coagulation of the suspended material. Coagulation gradually takes place as the polyphosphate reverts to orthophosphate in the presence of water, thus precipitating the alkaline earth metals. Versene and its sodium salts differ in that they react directly with a metal cation and form a stable soluble reaction product, i.e., the chelate of the metal. Such chelates are quite stable over a wide pH range. In most cases, they are not decomposed by either weakly acid or highly alkaline solutions. Temperature, time and addition of common precipitation agents have no effect on metal ions

SOLVENT ACTION OF VERSENE ON URINARY CALCULI

321

so chelated. This metal chelate, being a product of a definite chemical reaction, requires a specific amount of chelating agent to obtain the desired result. Insufficient amounts leave some unreacted metal ions and such residual ions can be precipitated. It is clear from the facts presented that chelating agents find their greatest use in the treatment of alkali metal salts since the chelating power increases as the pH rises. Applications of this unusual chelating power of versene has been made in the following commercial, industrial and hygienic fields. 1) Its most obvious use is in the general field of water softening. 2) This function may be utilized in detergent mixtures, clarification of soap, and removal of trace metal ions, which act as pro-oxidants of soaps, fats and oils. 3) It has a distinct use in the hydrolysis and dissolving of proteins, metal proteinates or other metal compounds in which the metal exists either as a or complex salt. 4) The "reserve" alkalinity of tetra sodium salt of versene is very useful in certain chemical reactions, (a) direct addition of this salt to a fat results in saponification and formation of a soap containing the alkali metal salt of versene, (b) similar reactions may be carried out with numerous organic and inorganic compounds to produce solutions with controlled composition and 5) This compound has been used in different industries, i.e., rubber (natural and synthetic), dye, textile, flotation, separation of metals, paints, varnishes, stabilizers for plastics, etc. 'l'OXICITY OF VERSENE AND ITS SAL'l'S

An increasing amount of studies on the toxicity of Versene are becoming available. From the evidence at hand, versene is no more toxic than any other alkaline solution of equal pH. In regard to pH, the tetra sodium salt has a relatively high pH, tri-sodium and disodium salts have correspondingly lower depending on the number and degree of ionization of the hydrogen atoms. The following reports are significant: 1) The Bersworth Chemical Co. has not observed any ill effects in the plant personnel over a 10 year period during Trhich time intensive production of very large quantities of versenc and its derivatives, ranging in pH from 3 to was carried out. 2) The same compounds have been used in industry and research in large amounts and in formulated proprietary products without complaint of any kind. 3) Patch tests with versene at pH 7 and 8.5 applied at 0.50 per cent concentration shows no irritating effects. Intradermal injections of 2 per cent neutral sodium salt of versene and castile soap in physiological saline solution ,vas not injurious to test animals. Control animals injected ·with soap and saline solution only showed that the triple combination is no more irritating than the control. 5) Oral feeding tests have been carried out by Dr. Martin Rubin, Georgetmvn Medical School, Washington, D. C. Versene was fed in drinking water to large

322

BENJAMIN S. ABESHOUSE AND TOBIAS WEINBERG

groups of rats for a period of 1 year at dose levels of approximately 2 to 5 per cent of total food and water intake, i.e., 12.5 mg. of versene per ml. All test rats gained weight with no fatalities or signs of toxic effect. These experiments are being continued for longer periods to establish the toxicity limits under conditions more closely conforming to potential utilizations in food and beverages. 6) The effect of parenteral injection of tetra sodium salt of versene was studied by Dykerkoff and coworkers who found that parenteral injection of 80 mg./kg. produced no toxic effects in rabbits. Tetany was noted at 125 mg./kg. but recovery followed injection of 7 mg. calcium in the form of calcium chloride. Ru bin found that versene is absorbed by the systemic route and exerts its calcium depressing action rapidly in the case of intraperitoneal injection being 100 per cent fatal at a dose of 100 mg./kg. Versene is absorbed more slowly from intramuscular sites and least rapidly from subcutaneous sites. 7) Rubin studied the effects of the intravenous administration of purified versene (100 mg./kg., pH 7.2) in rabbits. When the solution was injected rapidly, i.e., within 15 seconds, it was found that at lethal doses of versene the blood level falls precipitously and the animals die in convulsive tetany. The results obtained after the rapid injection are indicated in the following table. Dose, mg./kg,

% Survival

100 50 25 15 12.5 10 5

0 0 10 60 80 100 100

When the intravenous injection was extended over a period of 10 minutes at a dose level of 50 mg./kg., the survival rate was 100 per cent with little change in the available calcium levels. Administration of intravenous infusion over a period of 3 hours allowed the introduction of 2000 mg./kg. of versene before the available calcium was depressed to a level incompatible with life. When versene was administered intravenously to rabbits in 5 mg./kg. daily doses for 30 to 60 days, there were no significant changes in the blood calcium levels, no evidence of cumulative toxicity and no histologic evidence of damage to the liver, kidney, adrenal or other organs. Rubin found that the calcium complex of versene is virtually nontoxic by any route of administration. Dosages of 750 mg./kg. injected intravenously to rabbits showed 100 per cent survival. Rubin concluded that the toxicity of versene to animals is due to its function in binding calcium, and that its acute toxicity may be minimized by the slow administration of the agent which allows for replacement of calcium in the blood circulation by the labile reserves of the animal. He is of the opinion that versene will be found acceptible for human use as an adjunct in the processing of drugs, foods and beverages. EXPERIMENTAL STUDY

Any experimental investigation of the physical properties and chemical composition of urinary calculi entails many complex and time consuming studies.

SOLVENT ACTION OF VERSENE ON URINARY CALCULI

323

The present study of solvent action of versene upon urinary calculi may be divided as follows : 1) Study of calculi. a. identification, i.e., chemical composition. b. preparation, i.e., dehydration, sawing, sealing. 2) Solvent action of versene upon calculi in vitro. a. preparation of versene solutions. b. special irrigation apparatus for solubility studies. 3) Study of toxic action of various concentrations of versene. a. effect on the lining membrane of renal pelvis, ureter and bladder. 4) Solvent action of versene on calculi in vivo. a. stones placed in dilated pelvis of experimentally produced hydronephrosis. TABLE

2. Crystalline components of urinary calculi as determined by spectographic and x-ray diffraction methods by Prien and Frondel FORMULA

CRYSTALLINE COMPONENTS

Calcium oxalate monohydrate .... . Calcium oxalate dihydrate ....... . Magnesium ammonium phosphate hexahydrate ................... . Carbonate-apatite ................ . Hydroxyl-a pa ti te ................. . Calcium hydrogen phosphate dihydrate ........................ . Uric acid ........................ . Cystine .......................... . Sodium acid urate ................ . *Tricalcium phosphate ........... . tind.igo ........................ . tXanthine ....................... .

MINERALOGICAL NAME

CaC204H20 CaC20,2H20

Whewellite Weddellite

MgNH,PO,,H 20 Ca1o(PO.C030H) ,OH2 Ca1o(PO,) ,(OH)z

Stmvite Carbonate-apatite Hydroxyl-apatite

CaHP0.2H20 C.H,N,03 SCH2CH(NH,)-COOH NaHC.H203N,H 20 Ca,(PO,), C1,H10N202 C,H.N,02

Brushite

Whitlockite

*Not found but identity established. but identity acceptable.

t Not found

Study of calculi. It is readily apparent that the dissolution of a specific calculus is directly dependent upon its chemical composition. Various methods of determining the qualitative composition of calculi have been devised. We are greatly indebted to Jensen (1939) and Prien (1941) for their application of modern analytical techniques of mineralogy to the identification of the crystalline components of calculi. Utilizing the polarizing (petrographic) microscope and x-ray diffraction techniques, in a study of 700 calculi, Prien and Frondel found only 9 distinct crystalline substances to be present in calculi as indicated in table 2. They showed that many supposed crystalline components of calculi recorded in the urological literature by many investigators are actually nonexistent as determined by modern physical methods of identification. Their studies served to emphasize the inadequacies of the qualitative chemical analysis routinely employed in most clinics throughout the world. They also showed that the wide

324

BENJAMIN S . ABESHOUSE AND TOBIAS WEINBERG

discrepancies exist between the results of analysis of calculi by modern physical methods and those obtained by simple qualitative chemical analysis and attribute these differences to 1) indeterminate nature of the reactions in many of the procedures used in chemical tests, 2) invalid reactions resulting from interference by organic substances of unknown composition, 3) the complex chemical structure of many crystalloids present in calculi, particularly the phosphates and oxalates, and 4) the small size of many calculi often prevents a complete chemical examination. The one and only objection to the routine use of polarizing microscopic and x-ray diffraction technique in the analysis of stones is that these TABLE 3. Classification of 124 calculi studied by the authors A- Chemical composition determined by physical methods by Prien B-Chemical composition determined by qualitative chemical methods by authors CHEl{ICAL COMPOSITION OP' CALCULI

1. Uric acid . . ... ..... . . . .. . . ...... . .. . .. .. ... . ... . ... . .. . . .. . 2. Uric acid, calcium oxalate . .. . . .. . . ... . .. . . ...... . . ... .... . 3. Uric acid, calcium, magnesium, ammonium oxalate, xanthin .. .............................. . ..... .......... . 4. Uric acid, calcium, magnesium, oxalate, phosphate .... . . . . . 5. Uric acid, calcium magnesium, phosphate .. . .. . . ........ . . . 6. Cystine ...... . . .... ... . . . . .. . . . . . ... .. .... . . ... . . ... ..... . 7. Cystine, apatite .. .. . ... .. .. .. . .... . . . .. . . . . .. ........ . . . . . 8. Calcium oxalate ..... ... ..... . . . . . . . . . . ... . .. . ... ..... . .. . 9. Calcium, ammonium oxalate . .. . .. .... .. . .. .. .. .. ... . .. . .. . 10. Calcium, magnesium oxalate ..... . . . .... . ... . . . . . . . . .. .. . . . 11. Calcium, magnesium, oxalate phosphate .. .. . ........ . . . . . . 12. Calcium, magnesium, oxalate, sulfate . . . .. . . .. . ... .. . . . . . . . 13. Calcium carbonate .... . ..... . .. . .. .. .... .. .. . .. .. . . . . .... . 14. Calcium ammonium carbonate . .. . . . . ... . . . . . . . . . . . . . . . ... . 15. Carbonate apatite . . .. .... . .... .. . ... ..... . . . . . ...... . . . .. . 16. Calcium monohydrate, apatite ..... . ... . . . . . . .. . ...... ... . 10. Calcium, magnesium, carbonate, phosphate . . .. . .... . ..... . 18. Calcium, magnesium, ammonium, carbonate, phosphate and oxalate ....... . ... . . .. ..... ... ..... . ... .. ....... . .. .

NO, OF CALCULI

3A 1A lB

IA IA IA IA 6A IA

lB lB

IA IA 2A 1B

lA lA lA 2A 96 B

24 A 100 B

methods require costly apparatus and specially trained technicians who are not available in every hospital and city. The authors are cognizant of the failings and inadequacies of qualitative chemical analysis of calculi. However since the necessary apparatus and technicians to carry out the physical methods of analysis were not available in Baltimore, we were forced to reply on qualitative chemical tests to determine the composition of the calculi employed in this study with the exception of 24 calculi contributed by Dr. E. L. Prien. In a study of 700 calculi, Prien and Frondel found only 9 distinct crystalline substances present.

SOLVENT ACTION OF VE RSENE ON URINARY CALCULI

325

Identification of calculi (table 3): In the series of 124 calculi studied by the authors, the chemical composition of 24 stones had been previously determined by Prien by physical methods. The chemical composition of the remainder (which were taken from the collection of calculi in the Pathological Department of Sinai Hospital or from the Brady Urological Institute of the Johns Hopkins Hospital through the courtesy of Drs. W. W. Scott and W. E. Goodwin) was determined by employing the schema for qualitative analysis proposed by Scherer, Claffey and Keyser in 1945 with some minor modifications. In this experimental study each calculus was sawed into two parts and the saw dust used for analysis as a representative sample of all material in the calculus. In the case of a concentric laminated calculi, a sample was obtained from each layer separately, powdered in a mortar and analysed separately. Small calculi were powdered in toto and analysed. Each stone was tested for the presence of organic matter and then subjected to the following individual tests, viz., uric acid and urates, xanthine, cystine, urostealith. The following tests were also performed; viz., calcium, magnesium, ammonium, phosphates, carbonates and oxalates. Preparation of the calculus: After the qualitative analysis of the stone was completed, each half of the stone was dried at 105 C for ½to 1 hours, the stone being weighed before and after drying. The freshly cut surface of each part was coated with a thin layer of DeKhotinsky cement in order to prevent the penetration of the solvent through the cut surface. Only in this manner could the conditions prevailing in the uncut stone be duplicated. The stone was again weighed after application of the cement. The effect of dehydration upon the weight of the calculi was studied. Five calculi were placed in drying oven at 105 C for varying periods of time. It was found that after ½hour there was an average loss of 10 per cent; after 1 hour an average loss of 15 per cent and after 1½ hours an average loss of 16 per cent. No further appreciable loss was observed after 2 hours and up to 6 hours. Study of solvent action of versene upon the calculus (fig. 1): A special apparatus was employed for continuous irrigation of the stone with the versene solution. This apparatus was designed by Mr. Thomas Hofmaster, research laboratory technician at the Sinai Hospital, and is a simple and efficient apparatus which can easily be constructed in any laboratory. The apparatus consists of : A. Reservoir containing versene solution is a 500 cc flask with a narrow mouth occluded by a rubber stopper with three holes through which three (¼ in.) glass tubes are inserted. One tube transmits the versene to the irrigation apparatus by gravity; a second tube is connected to a vacuum apparatus, (V), not shown in drawing, and the third tube draws irrigating fluid up from the collecting receptacle back into the reservoir. B. Clamp used to regulate the rate of flow (10 cc per minute). C. Murphy drip mechanism. D. Stone chamber consists of a discarded stainless steel blood filter and has numerous fine openings to permit constant contact of versene with the stone. E. The prepared stone.

326

BENJAMIN S. ABESHOUSE AND TOBIAS WEINBERG

F. Thermoregulator to control the temperature of the water bath which is usually maintained at 37.4 C. G. A long glass tubing (¼ in.) diameter whose lower end is bent to hold the stone chamber in position within the irrigation chamber, thus permitting continuous irrigation of the stone. H . An electric hot plate on which is placed water bath (500 cc pyrex flask) for immersion of the irrigation chamber. J. Irrigation chamber constructed in the laboratory of large glass tube with closed end measuring 10 cm. in length by 3 cm. in diameter. An outlet tube is

A

A-RESERVOIR (300CC)

B---i:==:

8-CLAMP C-MURPHY DRIP 0 -STAINLESS STEEL STONE CHAMBER E- STONE F-THERMO REGULATOR G-1/4" GLASS TUBE H-HOTPLATE J - IRRIGATING CHAMBER K- FEEDING CHAMBER (500 CC.) V-VACUUM W-WATER BATH

APPARATUS FOR THE IRRIGATION OF CALCULI .WITH VERSENE

Fig. 1. Apparatus for irrigation of stones with versene

fused in the midportion to permit the escape of the irrigating fluid into receiving receptacle which is a glass beaker 100 cc from which the fluid is drawn back by vacuum into the reservoir to be used over again. K. Glass feeding chamber-composed of a long glass chamber (300 cc) with tapering outlet which is connected by a rubber tubing to the Murphy drip mechanism. After the desired period of irrigation with versene solution the calculi were washed with distilled water until free from versene solution and salts. The calculus was dried again at 105 C for ½to 6 hours and the loss in weight was recorded as per cent dissolution. Preparation of versene solution: The versene solution was prepared in 0.5, 1,

SOLVENT ACTION OF VERSENE ON URINARY CALCULI

327

2, 5, 10, 12.5, 15, 20 and 50 per cent solutions by adding the required amount of purified versene powder to 100 cc of sterile distilled water. To each solution, enough sodium hydroxide (NaOH-10 per cent solution) was added to bring the pH to 7.2 as determined by Lamotte pH indicators or by electrometric methods. Accurate measurment of the pH should be made with a pH meter utilizing a glass electrode when this apparatus is available. This method was employed for the preparation of the solutions in the in vivo studies. Determination of pH by special litmus paper is not sufficiently accurate to warrant its use. The last step is sterilization of the alkaline versene solution by autoclave at the standard pressures used in the operating room. Preliminary study of solvent action of versene. The preliminary study consisted of testing the solubility of several different calculi of various chemical compos % DISSOLUTION

60%

IMMERSION OF STONE IN 5eo. VERSENE(l0%)FOR 6 HRS.

50%

40%

30%

20%

10%

0% . ..__ _~O_,.¾_ __ URATE

URIC ACID

(;'!'STINE

CALCIUM OXALATE

ALKALINE

CALCIUM

EARTH CARBONATE PHOSPHATES

1Frn. 2. Illustrating amount of dissolution of urinary calculi following immersion of different calculi in 5 cc versene (10 per cent) for 6 hours.

tions in versene solutions of different concentrations, i.e., 0.5, 1, 2, 5, 10, 20 and 50 per cent. A small calculi of known weight was placed in 5 cc of each of the different concentrations of versene solution for 1 to 24 hours. The calculi were weighed after such treatment and the difference in weight recorded as per cent of dissolution. An analysis of the data (fig. 2) of this specific study revealed: 1) The most effective solvent action is observed with the 10 per cent solution of versene. The chelating action of 10 per cent versene was essentially the same as 15 per cent and 20 per cent versene solution. 2) Uric acid and/ or urate stones are not affected by versene solution in various concentrations. 3) Calcium carbonate calculi were dissolved more readily than other calculi, i.e., 51.5 per cent dissolved in 6 hours with 10 per cent versene solution. 4) Mixed alkaline earth phosphatic calculi had a high ratio of solubility, i.e., about 45 to 50 per cent dissolution in 6 hours with 10 per cent versene solution.

328

BENJAMIN S. ABESHOUSE AND TOBIAS WEINBERG

5) Calcium oxalate in pure or combined forms are less readily dissolved, i.e., 31.1 per cent in 6 hours with 10 per cent versene solution. 6) Cystine calculi exhibited a 15 per cent dissolution in 6 hours. Experimental study of the dissolution of calculi by irrigation with versene solution. This study consisted of subjecting 120 different calculi to irrigation with different concentrations of versene over varying periods of time. The majority of the calculi studied were composed of calcium, ammonium, magnesium, phosphate, carbonate and oxalate which represents the usual type of mixed stone encountered in our practice as well as in the practice of others in this country. The calculi were carefully dried and weighed before and after irrigation with versene solution and the loss of weight recorded as per cent dissolution. RATE OF DISSOLUTION OF URINARY CALCULI FOLLOWING IRRIGATION WITH 10 % VERSENE 10011:f 90%

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8011:

/

7011:

/ 6011:

40%

3011: 20%

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10%

o11:=-----+---2!-----:!-3---4.J__

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TIME OF EXPOSURE. HOURS A.AMMONIA , CALCIUM CARBONATE ·P66II B .AMMONIA, CALCIUM CARBONATE PHOSPHATE OXALATE MAGNESIUM C .CALCIUM CARBONATE .

FIG. 3. Showing rate of dissolution of calculi followi ng irrigation with 10 per cent versene over a period of 24 hours.

Analysis of the results of these experiments revealed: 1) A very high rate of dissolution was obtained with 10 per cent versene solution which only differed by ±2 per cent from a 15 per cent or 20 per cent versene solution. 2) There was a progressive increase in per cent of dissolution of such calculi following continuous irrigation with 10 per cent versene solution. The following results represent the average loss (per cent of dissolution) at the end of specific time intervals, viz., 30 minutes .... . . . ..... . . .... .. . . . . . .. . . .. ... . .. ... . . . . ... . .. . . ... . . ... 1 hour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 hours. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 hours ... . . ..... . . . . ..... ... .. . ..... . . ...... . .. . .. . . .. . .... . .. .... .. 18 hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 hours. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 hours .. ..... .... .. ....... ... . . . . . . .. .... .. . . .... .. .. . . .. . .........

10-12% 20-25% 35-45% 50-60% 60-65% 78-80% 88-100%

The graph (fig. 3) shows the results of irrigation with 10 per cent versene over a period of 24 to 48 hours.

SOLVENT ACTION OF VERSENE ON URINARY CALCULI

329

Experimental study of toxic action of 10% versene solution upon the mucosa of the renal pelvis, ureter and bladder. A. Continuous irrigation of the bladder of 4 rabbits with 10 per cent versene (buffered to pH of 7.2 with 10% NaOH) through a No. 16 double irrigating Foley catheter over a period of 4 hours disclosed moderately severe irritative changes on gross examination. Histological studies also revealed superficial and deep changes in the mucosa or submucosa. B. Irrigation of the renal pelvis of 6 dogs with 10 per cent versene revealed moderately severe irritative action on gross and microscopic examination. This study was carried out in three experiments, viz., 1) Retrograde catheterization of the normal ureter in large dogs (4 F catheter). 2) Retrograde catheterization of the normal undilated pelvis and ureter through a cutaneous ureterostomy (6 F double lumen catheter) . 3) Retrograde catheterization of the dilated renal pelvis and ureter, i.e., resulting from partial constriction of the lower ureter by ligatures or surrounding the lower end of ureter. A 6 F double lumen catheter passed up through the ureterocutaneous opening. Practically the same percentage of dissolution was obtained with weaker solutions of versene, i.e., 7.5, 5 and 3 per cent with a pH of 7.2, with correspondingly less irritation. The degree of irritation was decidedly less when NaHC03 was employed instead of N aOH as the alkalizing agent. Experimental study of the dissolution of calculi "in vivo". Unilateral dilatation of the pelvis and ureter was produced 1) by partial constriction of the ureter by ligature or 2) by placing a one inch piece of polyethylene around the lower ureter. After a period of 14 to 21 days, the renal pelvis was exposed and incised and a small calculus i.e., mixed alkaline earth, phosphate, oxalate, carbonate, was placed within the pelvis which was carefully resutured to preserve its integrity. The ureter was severed in its lowest portion, i.e., close to the site of constriction and was transplanted to the loin. A No. 6 or No. 9 double lumen catheter was passed up the ureter into the pelvis and continuous irrigation of the pelvis with 10 per cent versene was performed for 4 to 6 hours. It was observed that 25-30 per cent dissolution of the calculus was obtained after irrigation for 4 to 6 hours but was accompanied by severe chemical irritation of the pelvis and a necrotizing papillitis. It is interesting to note that essentially the same percentage of dissolution was obtained with a 7.5, 5, and 3 per cent solution of versene (pH 7.2 with NaOH or NaHC0 3) with a proportionate decrease in the degree of irritation. When a 3 per cent solution of versene (pH 7.2 with NaHC03 ) was employed, the degree of irritation of the renal pelvic epithelium was minimum and in several cases was entirely absent. SUMMARY

The authors have attacked the problem of the dissolution of calculi in vitro by employing for the first time the chemical process of chelation and a new chelating agent, i.e., versene. Our studies indicate that 10 per cent versene is a particularly effective solvent in vitro for the following types of calculi, viz, calcium carbonate and mixed alkaline earth phosphates. It is also effective to a lesser degree in the dissolution of calcium oxalate calculi in pure or combined

330

BENJAMIN S. ABESHOUSE AND TOBIAS WEINBERG

forms. It is surprising to note that cystine calculi in pure or combined forms are also dissolved by 10 per cent versene solution. Uric acid and urate calculi are not affected by versene solution in various concentrations. Experimental studies were undertaken to determine the toxic action of 10 per cent versene (pH 7.2) upon the lining membrane of the renal pelvis, ureter and bladder of dogs and rabbits and it was found that continuous irrigation of these structures for 4 to 6 hours produced demonstrable irritative action of the epithelial lining of the bladder, ureter and pelvis. Experimental studies in dogs of the dissolution of calculi in vivo i.e., retrograde irrigation of hydronephrotic pelvic containing calculi reveal that approximately 25 per cent dissolution of alkaline earth phosphatic calculi after 4 hours irrigation with 10 per cent versene but was accompanied by irritation of the renal pelvis and papillae. The authors are continuing their studies of the toxic effects of versene and its salts upon the bladder and renal epithelium. This study is still in progress but it does appear that weaker solutions of versene acid and its various salts, i.e., 3, 5, and 7.5 per cent are decidedly less irritative. Studies are also in progress to determine the influence and relation of alterations in the pH of the various versene solutions, i.e., utilizing various inorganic and organic acidifying and alkalinizing agents, upon the toxic and irritative properties of the solution. We believe that these studies will reveal the ideal strength and pH of versene to insure its chelating action without irritative action on the urinary organs. The authors are of the opinion that this new chelating agent offers definite promise as a solvent for urinary calculi in humans. Clinical trials will be undertaken as soon as the appropriate non toxic and non irritating solution of versene is determined and substantiated by animal experiments. A subsequent report of these results will be presented at a later date. This study has also prompted the authors to investigate the efficacy of other organic sequestering and chelating compounds in the dissolution of urinary calculi and the results of this study will be reported later. The authors wish to express their sincere thanks to Drs. E. L. Prien, W. W. Scott and W. Goodwin who contributed some of the stones used in this study, and to Dr. Mano Golden, Thomas Hofmaster and Melvin Fuld for their invaluable technical assistance.

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