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Experimental Observations on the Pathogenesis of Urinary Calculi
Vol. 95, May Printed in U.S.A..
TnE JorRNAL OF UROLOGY
Copyright© 1966 by The Williams & Wilkins Co.
EXPERIMENTAL OBSERVATIONS ON THE PATHOGENESIS OF URINARY CALCULI C. W. VERMEULEN, J.E. ELLIS
"ND
TE-CHIN HSU*
From the Department of Surgery, University of Chicago, Chicago, Illinois
Ebstein and Nicolaier, in a classical monograph on experimental urolithiasis published in 1891, arrived at a concept of the pathogenesis of stone formation in which the organic matrix was considered as a sine qua non for the process. 1 They believed that the mechanism for stone growth began by some kind of combination between an albuminous niatrix material and certain dissolved minerals. Thereafter the mineral crystallized out of solution so as to mineralize the matrix ground substance in which they had been dissolved. Subsequently other investigators have held somewhat similar views and it has become widely accepted that the stone matrix occupies an extremely important and vital position in the stone-forming mechanism. 2 ,3 In sharp contrast, recent work in our laboratory has led to an hypothesis in which stone matrix is considered not as a crucial element, but rather as an incidental inclusion in stones. This hypothesis arose as the result of experiments in which an attempt was made to imitate stone formation in vitro using a technique reported in detail elsewhere. 4 • 5 Essentially, the method consists in repeated controlled crystallization of stone minerals in a medium of urine using a system where a wire loop serves as a nidus for minAccepted for publication July 2, 1965. Read at annual meeting of American Urological Association, Inc., New Orleans, Louisiana, May 10-13, 1965.
era! deposition" The system can be modified in various ways so as to yield deposits of most of the stone-forming minerals and also of uric acid and cystine. The deposits, or artificial concretions, often assume many of the structural characteristics of authentic calculi. Surprisingly, they also contain a matrix-like component. Further experiments have shown that the matrix arises by incorporation of non-dialyzable urinary constituents into the developing concretion, for if a medium of urine ultrafiltrate is used in place of whole urine, no such matrix component is present. Hence the concretion matrix, though always present in objects prepared in whole urine, is not a necessary constituent. Instead it might be considered as an incidental inclusion that results from surface adsorption of protein upon the crystallizing mineral, a phenomenon well-known in crystal theory. If these in vitro results are extrapolated to the formation of true stones, attempts to understand the pathogenesis of calculi would focus not upon some cryptic vital matrix activity but rather upon crystallization per se. The purpose of the present paper is to elaborate upon this view and, with the aid of further in vitro observations, to develop a general hypothesis of the stone process in terms of crystallization. Some illustrative in vivo experiments are also included.
This investigation was supported by grant AM 475 from the National Institute of Arthritis and
Metabolic Diseases, National Institutes of Health. * Present address: Department of Urology, National Taiwan University, Taipei, Taiwan. 1 Ebstein, W. and Nicolaier, A.: Experimentelle Erzeugung von Harnsteinen. Wiesbaden: J. F. Bergmann, 1891. 2 Boyce, W. H., and King, J. S., Jr.: Crystalmatrix interrelations in calculi. J. Urol., 81: 351, 1959. 3 Cornelius, C. E.: Studies on ovine urinary biocolloids and phosphatic calculosis. Ann. N.Y. Acad Sci., 104: 638, 1963. 4 Vermeulen, C. W., Lyon, E. S. and Gill, W. B.: Artificial urinary concretions. Invest. U rol., 1:
370, 1964. 5 Fried, F. A. and Vermeulen, C. W.: Artificial uric acid concretions and observations on uric acid solubility and supersaturation. Invest. Urol., 2: 131, 1964.
681
IN VITRO EXPERIMENTS
If we are to explain stone formation by means of crystallization phenomena, it will be helpful first to consider certain fundamentals of crystallization, in particular the concepts of supersaturation and nucleation. Both of these terms are occasionally used in an imprecise n1anner in discussion of stone mechanisms. Supersaturation. It is well known that the stone minerals are generally more soluble in urine than in water even at an equivalent pH. JVIany other urinary components-both specific (e.g. citrate) and non-specific-enhance the solubilizing power of urine. However, the supersolubility in urine
682
VERMEULEN, ELLIS AND HSU
SUPERSATURATED
SATURATED
n
acidify ~
D
Uric a. sol 'n
LJ
~
further acidif.
.
spontaneous nucleation
. .
extraneous
nucleation
evap,
t
D
seed with crystal
SUPERSATURATED
______,.
~
primitive ~
polycryst. mass
re-nucleation at crystal
FIG. 1. Schematic illustration of supersatura-
tion and nucleation using uric acid as solute and water as solvent. See text for details.
compared to water is not to be confused with supersaturation. In true supersaturation the amount of dissolved solute is actually greater than is present in a saturated solution in the identical solvent, a condition that can be induced only in special ways. Figure 1 illu,strates 2 methods, both starting from a solution already saturated. In one, the rnlvent power of the medium is slightly reduced by adding acid to decrease the solubility of uric acid; in the other, the nature of the solvent it,elf i, unchanged, but its volume is somewhat reduced by evaporation. If either of these operations is carefully performed and not too drastic in extent, precipitation may be much delayed or fail to occur entirely, as if the solute were reluctant to enter the solid state. Meanwhile, the solution obviously becomes truly super,mturated. It fhould be noted that both methods have their counterparts in the normal processes of urine forrn.ation, for a very dilute glomerular filtrate is gradually concentrated by reabsorption of water and at the same time modified in a variety of other ways. Therefore, it would be reasonable to find that some of the poorly soluble materials ultimately reach a supersaturated condition and such, indeed, is often the case. Nucleation. The state of supersaturation is clearly an unstable condition and when it becomes excessive, precipitation will eventually develop. But prior to actual precipitation, the phenomenon of nucleation is necessary. By nucleation is meant the appearance within the solution of sub-microscopic aggregates of molecules (uric acid molecules in the illustration of fig. 1) where the aggregates satisfy 2 criteria:
FIG. 2. Development of adherent polycrystalline mass in supersaturated solution of copper sulfate in water. A, original seed crystal of copper sulfate. B, crystal has grown but new crystals have also appeared. C, successive generations of crystals have led to large mass. (Measuring bar in C represents 1 cm. But A and B are magnified 5 times greater than C.)
1) the molecular collection, though formed by random molecular movements, must happen to have its molecular unit, arranged in the proper crystal lattice ori2ntation for the particular substance and 2) the,e randomly formed aggregates mmt be of a certain critical size, for if too small they immediately disperse and dissolve back into true solution. Only when aggregates appear which are larger than the critical size will they succeed in growing by acting like miniature seed crystals. Thus, the reason why a supersaturated solution may temporarily remain in a state of apparent stability is that nuclei, as so defined, have not appeared. But as soon a3 nuclei of the critical size are present, they grow rapidly and produce a visible precipitate until, eventually, the state of supersaturation is relieved. Naturally, nucleation becomes more likely as the degree of supersaturation increase3; and if nucleation occurs simply became supersaturation is extreme, it may be designated as of the spontaneous type. However, the degree of supersaturation is not the sole determinant; mere agitation, a scratch on the inside of a glass container, dust particle,, debris, and foreign surfaces may induce nucleation that might otherwise not appear spontaneously. This type of nucleation might be termed extraneom. Additionally, figure 1 shows a special circumstance where new nucleation sites formed at or near the surface of a pre-existent crystal. Though these new sites are often swallowed up by the larger crystal, it is possible for them. to develop into new crystals
PATHOGENESIS OF URINARY CALCULI
683
FIG. 3. Photomicrographs show production of cystine polycrystalline mass in supersaturnted water solution, beginning with single crystal (A) attached to fine glass filament. D (at much lower magnification) shows not only further growth but also reworking, with crystals fusing toward glassy mass. (Magnifications: Measuring bar in D represents 1 mm. But relative to D, A is magnified 40X, B, 30X and C, 20X.)
FIG. 4. Habit modification in artificial concretions of calcium phosphate. A, formed in medium of water, yielding loose powdery deposit. B, formed in urea-salt solution, showing hard concretion with leathery surface. (Measuring bar represents 1 mm.)
684
VERMEULEN, ELLIS AND HSU
FIG. 5. Habit modification of calcium phosphate concretions. A, concretions of adherent spherules formed in medium of urea-salt solution. B, same medium except that, it also contained 10 per cent mg. Concretion is hard smooth crust. (Measuring bar represents 1 mm.)
now attached to the old. In consequence, a polycrystalline mass results, held together by crystal forces alone. Production of simple polycrystalline masses. Figures 2 and 3 illustrate crystal masses formed with substances which tend to produce crystals sufficiently large for easy viewing. In one instance a crystal of copper sulfate was suspended in a supersaturated water solution (fig. 2). At first the crystal simply increased in size but with slight fluctuations in the degree of supersaturation, new crystals soon appeared on its surface. Successive generations followed and eventually a large hard adherent polycrystalline mass developed. Similarly, though at a micro level, figure 3 shows the production of a polycrystalline mass of cystine. The cystine illustration also dern.onstrates, though imperfectly, another phenomenon which might be called "reworking" of the polycrystalline mass. After photographing the object as seen in figure 3,C it was repeatedly subjected to alternating periods of slight undersaturation and supersaturation. As a result, the individual crystals are being reworked, fusing together and producing larger masses with indistinct outlines (fig. 3,D). If reworking were carried to con1pletion, the whole object could become an irregular glassy deposit somewhat like ice.
Artificial concretions and habit modification. The technique previously mentioned for producing artificial urinary concretions can be used with media of simple water solutions as well as with urine. The simpler medium then permits more exact control of its composition and allows demonstration of the remarkable variation in the appearance of the concretion that may result from seemingly insignificant alterations in circumstances, a phenomenon that might be termed habit modification. The concretions shown in figures 4 and 5 are all of calcium phosphate (apatite-brushite composition) but they vary greatly in external appearance and texture. In figure 4,A the medium consisted of plain water and the deposit appeared as a loose fluffy material. However, if the water medium contained urea and NaCl (in concentrations approximating normal urine) a hard leathery object was produced quite different from the previous specimen (fig. 4,B). The object shown in figure 5,A was made in the same urea-salt medium but the ratio of Ca: P was altered somewhat and the concretion habit was changed, now appearing as a mass of fusing spherules. Finally, the effect of adding Mg ion (10 mg. per 100 rn.l.) to the urea-salt medium is shown in figure 5,B; here the deposit has taken on the forn1 of a very hard
685
PATHOGENESIS OF UhlNARY CALCULI
EFFECT OF "MATRIX" ON SPEED OF DISSOLUTION (Solvent O. 05N HCI) WITHOUT "MATRIX"
/WITH
"MA.TRIX"
20-
. Frn .. 6. Calcium phosphate concretions after d1ssolvmg away calcium phosphate. A, objects formed m 10 davs m urea-salt medium with chloroform as a11tibacterial agent. B, objects formed m 5 days aseptically in same medium. In both A and B bare wires (above) are obtained after dissolution. But when albumin, 100 mg. per cent, was added to the medium, matrix residues (below) are found. (Measuring bars represent 1 mm.)
crust. Similar experiments, both with calcium phosphate and other stone minerals, have repeatedly demonstrated the same extreme sensitivity to seemingly minor variations in either the medium or in technical details. Obviously the habit of polycrystalline masses is subject to pronounced alterations dependent upon the environment in which they develop. The condition seems to be somewhat analogous to habit modification of single crystals due to impurities in the crystallizing media, a condition of great interest to crystallographers in recent years. Protein adsorption and matrix. The occurrence of matrix-like components in artificial concretions produced in urine has already been mentioned. Similarly, a matrix is present in concretions developed in simple watery media containing a small amount of protein (fig. 6,A). However, previous publications have pointed out a possible objection to considering such matrices as really analogous to the matrix of true stones, for they could be merely factitious, the result of the bacteriostatic agents necessarily used in preparing the concretions. Ordinarily, either formaldehyde (0.2 gm. per 100 ml.) or a layer of chloroform beneath the urine or water medium has been used in the concretion technique in order to prevent bacterial growth. It is conceivable that the concretion matrix is the result of action by the antibacterial agent in denaturing and precipitating the protein. If so, the supposition that stone matrix arises simply by surface adsorption of unmodified protein would be illusory. Therefore,
HOURS
Frn. 7. Comparison of dissolution speeds of 2 calcium phosphate concretions that were identical in_ weight (58 mg.) and texture, one with and one without an albumin matrix. Concretions suspended and rotated in 60 ml. of 0.05 N HCl as solvent. Increasing concentrations of calcium reflect speed of dissolution. Retarding action of "matrix" is shown.
the experiment illustrated in figure 6,B is significant in resolving the doubt, for here sterility was maintained without using any antibacterial agent, and though the experiment was only of 5 days' duration, a definite matrix was obtained. Thus the matrix-like residue cannot be considered as a simple artifact. Impedence to dissolution by matrix. When concretions with and without matrix are subjected to solvents, it has been observed repeatedly that those with a matrix tend to dissolve more slowly. Figure 7 illustrates the result of one such experiment in which calcium phosphate concretions, identical in weight and indistinguishable in structure, were suspended and continuously rotated in a solvent consisting of 0.05 N HCl. As dissolution progressed, the calcium content of the solvent increased dependent upon the speed of dissolution. It is evident that the concretion without a matrix dissolved considerably more rapidly. Repeatedly, similar tests with other concretions have yielded similar findings. Perhaps an important implication can be derived from these observations if the stone-forming reaction is really a reversible one. In that case a stone would begin to dissolve if the urine concentration fell below the level of saturation. However, the matrix would remain behind to form a barrier to rapid penetration of the unsaturated urine. As a result, dissolution ·would be retarded.
686
VERMEULEN, ELLIS AND HSU
_FIG. 8. A and Bi views of oxamide spherulites formed in urine as seen through polarizing microscope without (A). and with (B) crossed polars. ~OX They consist of spherical clusters of minute elongate cry~tals radially ar~anged .. Can~ D, photomicrograph~ (not polarizing microscope) of oxamide crystallizat10n to show _habit modific~t1on. _Fro1!1 water solut10n (C), blade and dendritic forms appear. When the_ ,ya~er sol~t1on also contams uric acid (5 mg. per cent), oxamide crystallization (D) becomes spheruhtic m habit. 40X. IN VIVO OBSERVATIONS
The purpose in this section is to illustrate in rats rnme of the crystallization phenomena previously described. To do so, an old technique for producing stones in animals was chosen in which oxamide (the diamide of oxalic acid) is administered in the food. The oxamide is excreted as such in the urine. It is a relatively insoluble substance and if given in sufficient amounts, the animals have a severe form of lithiasis. As shown originally by Ebstein and Nicolaier these stones are composed of oxamide and alrn contain a typical matri.'i:.i Indeed, the conclusions by these early workers on oxamide stones are important contributions to the concept that matrix is a necessity in the stone-forming process. Oxamide crystallization. When oxamide crystallizes from urine in either in vivo or in vitro experiments, it assumes a spherulitic form, an interesting type of crystallization seen with many substances, including some of those present in human calculi. When the oxamide spherulites
were examined with the polarizing microscope many elongate crystals all radiating from the center of the sphere were noted (fig. 8,A and B). Oxamide characteristically crystallizes from both urine and urine ultrafiltrates in the spherulitic form but from water it appears as small blades which may sometimes repeatedly branch to assume a dendritic form (fig. 8,C). However, spherulites are not invariable in urine, for if crystallization is sufficiently rapid, the blade and dendritic form appears. Contrariwise, spherulitic crystallization can occur from water solutions if certain impurities are also present. Figure 8,D shows the effect of an impurity consisting of a small amount of uric acid (5 mg. per 100 ml.) present in the crystallizing medium. Here, then, is an illustration of habit modification in oxamide crystallization. The early stone lesion. When oxamide is given to rats in high dosage (1.2 gm. per 100 gm. of diet) for 2 or 3 days, excessive supersaturation results in nucleation. In consequence, spherulites
PATHOGENESIS OF URINARY CALCULI
. FIG. 9. E_arly lesions of renal papillae in rats given oxamide. A, small spherulites have formed and are lined up in lumens of collecting ducts. B, stone formmg upon papillary tip exhibiting spheruhtic growth. Below stone has been teased from papilla. On its inner' surface finger-like proJ ect10ns are seen where stone extended into dilated ducts. (Measuring bars represent 1 mm.)
appear in the urine. Immediate examination of the fresh kidney with the dissecting microscope may reveal an interesting early lesion in the renal papilla. Figure 9,A shows large spherulites lined up in the lumens of some of the papillary ducts. A somewhat later le~ion is seen in figure 9,B where a minute stone has developed upon the papillary tip. vVhen the stone was teased from the papilla, its inner aspect showed projections which previously extended up into the ducts. One gets the impression that some of the larger spherulites were unable to pass through the openings of the ducts at the papillary tip and that the retained spherulites then acted as growth centers for development of a papillary stone or encrustation. If rats are maintained on this high dosage, death will occur within 14 days, but at autopsy papillary lesions are usually no longer found. Instead, calculi are present throughout the urinary system. Evidently, the initial papillary stones sloughed away from the papilla; and while most of the fragments were no doubt discharged with the urine, others were retained in various parts of the urinary system to act as further growth centers. The resultant stones still show evidence of spherulitic crystallization (fig. 10) but as new crystals are laid clown upon them, the spherulites grow and fuse into a unified mass. Experiment on supersaturation with and without nucleation. It has previously been shown that if oxamicle is given in an amount which, of itself, is insufficient to produce stones spontaneously, a
687
FIG. _10. Surface_ of oxamide stone. Spherulites of varymg size fusmg mto unified mass. (Measuring scale is in mm.)
foreign body implanted in the bladder will nevertheless result in oxamicle stone formation throughout the urinary system. 6 The experiment was interpreted as showing that the foreign body acted by inducing nucleation (extraneous type) when the urine was supersaturated, but not to the degree where nucleation would occur spontaneously. In the experiment reported in the present paper, nucleation is provoked in another way~ by initial administration of a shock nucleating close of oxamicle. Thus the animals receive no operation or surgical trauma to produce a local lesion. Instead, a preliminary high close of oxamide is used simply to initiate nucleation. Thereafter, treatment is continued with the lower dosage. In this way it might be possible to shavv the significance of nucleation as an essential factor in stone production without any operative insult to the urinary system whatever. Four groups of adult Holtzman female rats were used. One group, the primary control, received 0.3 gm. oxamicle per 100 gm. of Purina laboratory chow diet throughout the 4-week experiment. Another group was given the same dosage except that a nucleating shock close (1.2 gm. per 100 gm.) was used for the first 3 clays. Two secondary control groups were also included: in one of them, the shock dose was given for 4 clays and the 21 animals were sacrificed immediately thereafter and, in the other, the shock close was also given for 4 days but oxamicle was then completely 6 Gill, W. B. and Vermeulen, C. W.: Oxamide crystalluria and urolithiasis, rat and in vitro observations. Invest. Urol., 1: 339, 1964,
688
VEI::WEUL:EN, ELLIS AKD HSU
Fm. 11. Oxamide stones formed in control rats on low dose. Each white bar indicates 1 rat. Stones were found in only 1 animal; they are shown in their approximate original anatomical locations. White bars are 15 mm. long.
Fm. 12.0xamide stones formed in rats where high shock dose was used initially to induce nucleation followed by low dose used in control group. All rats showed stones distributed in various parts of urinary system.
discontinued. These 22 animals were sacrificed at 4 weeks. Comparison of stone growth. Of 20 rats in the primary control group (where the initial nucleating dose was not used) stones were found in only 1 rat at the end of the 4-week experiment (fig. 11). However, all 21 rats of the experimental group showed stones widely distributed throughout the urinary system (fig. 12). It is obvious that the initial heavy dosage had a profound effect. But were these stones already completely formed during the first 3 days, or did they continue to grow in the period on the lower dose? One of the secondary control groups (shock dose for 4 days, then sacrifice) provides an answer, for only the expected minute papillary lesions,
along with fine spherulitic sand, could be seen when the urinary systems were examined with the hand lens. Stones such as those shown in figure 12 were not observed. Clearly, then, marked stone growth continued during the 25 days when the rats received the low dose. The experiment illustrates that stone initiation requires both supersaturation and nucleation. The nuclei then grow into crystals or minute polycrystalline masses (spherulites when oxamide is used) and if, by chance, any of these embryonic stones are retained in the urinary system, they have an opportunity to grow-provided the urine is still supersaturated. However, if the urine is sufficiently undersaturated, the embryonic stones will dissolve or be discharged with the urine, as
PATHOGENESIS OF URINARY CALCULI concentration etc.
A
spontaneous
\
- - - - - - nuclearion
ISUPERSATURATION I
extraneous
~
I
/
NUCLEATION
I CRYSTALLIZATION I/
t
.1
\
nucleation
/
cc:1::tion
POLYCRYSTALLINE/ MASS habit modifiers
/
t
'
"reworking"
adsorbed ''matrix"
B
no "matrix"
REVERSIBILITY AND DISSOLUTION WHEN UNDERSATURATED
DISSOLVED STATE
-----'------"'
~
~
SOLID STATE
~
\ "matrix" adsorbate impedes dissolution
FIG. 13. A, schematic formulation of stone producti?n viewed as crystallization process. B, expression of possible significance of matrix in retarding dissolution. I
i i
"' , I
shown in the other secondary control group. Here (where only the shock dose was given followed by 24 days without oxamide) all the rats were completely free of stones or papillary tip lesions. DISCUSSION
It is self evident that stone production must be intimately linked with phenomena concerned with the insolubility of certain substances in urine. But a stone is not a mere precipitate. Stones have an organized wholeness, are sometimes bizarre in external structure (e.g. jack stones) and often have a complex internal architecture (with lamination, radial striation, etc.). It would seem, then, that insolubility and factors controlling it would be insufficient to explain such a complex thing as a stone, though perhaps adequate to account for crystalluria. Something more than mere precipitation seems to be required to produce a stone. And "something more" is available, for all stones have an organic matrix. In consequence, matrix is commonly thought of as a cement holding the mineral together, or as a skeleton accounting for the complexity of stone structure, or, again, as a ground substance into which the mineral is deposited under the guidance of the matrix. These concepts seem to offer a bridge over the gap between precipitation processes on the one hand, and stone on the other. In this paper, on the contrary, stone matrix is given no such vital role. Instead, matrix is considered as an incidental inclusion, a notion arising
689
primarily fron, previous work on artificial concretions. If true, then the role of matrix as a bridge between simple precipitation and the complex wholeness of stones is lost, and it becomes necessary to find another explanation to take its place. Perhaps crystallization itself can cover the conceptual gap when it is realized that the field of crystallization concerns far more than the production of a mere precipitate or the development of single crystals. An amateur excursion in the field appears to indicate that the special roles ordinarily given to matrix can be accounted for by crystallization processes themselves. The miscellaneous experiments and observations presented in this paper are intended to suggest how a theory of basic stone-forming processes can be built around conditions affecting crystallization by considering a stone as a polycrystalline mass. Sometimes the mass is composed of very large crystals, as in calcium oxalate dihydrate or cystine stones, but usually the crystals are very much smaller and more or less fused into a mass not obviously crystalline in nature. Schematically, some of the principal factors concerned in crystallization are given in figure 13, a formulation which may serve to correlate the various experiments that have been presented. In the section on in vitro experiments, some of the basic principles of supersaturation and nucleation were discussed in some detail. Simple illustrations of polycrystalline masses were also given to indicate that a matrix cement is quite unnecessary to hold the crystals together (as is also illustrated, of course, in ordinary rocks and minerals). Evidence was also presented to supplement previous reports showing that the existence of matrix could be accounted for by surface adsorption of non-dialyzable material that is invariably present in urine. Further, some in vitro studies suggest how the complexity of structure and the peculiar, seemingly non-crystalline, texture of stone material could be explained by reworking and habit modification of the polycrystalline mass. Lastly, an experiment was reported to show that a matrix of adsorbed protein, though it does not govern the laying down of mineral, still can perform a function by impeding dissolution ,vhen conditions for demineralization might otherwise be suitable. The experiments with oxamide are in vivo illustrations of this attitude toward stone forma-
690
VERMEULEN, ELLIS AND HSU
tion as a crystallization process. The pronounced tendency toward spherulitic crystallization is helpful here not only as a means of identifying the early stone lesions, but also as an example of habit modification. Further, reworking and fusion of the primary crystalline units are illustrated upon examining the developing stones. The necessity for nucleation, in addition to supersaturation, could also be demonstrated by using the technique of an initial shock dose followed by a prolonged period on a dosage that is insufficient, of itself, to produce calculi. We might therefore reconstruct the course of stone formation here as follows: 1) Oxamide, a relatively insoluble substance, leads to supersaturation; and, if supersaturation is sufficiently pronounced, results in spontaneous nucleation. Oxamide crystallization ensues, occurring as spherulites because of habit modifiers as yet unidentified. (Though the in vitro experiments show that uric acid is such a modifier, it must be remembered that uric acid does not occur in rat urine in very significant amounts.) 2) Most of the spherulites are discharged with the urine but some of these forming in the papillary ducts fail to pass through the papillary ostia. These act ar foci for further crystallization, again in the spherulitic form. 3) After the papillary lesions slough off, most of
the fragments are probably expelled with the urine but some remain behind, either because of their size or by pure chance, and act as giant nuclei for further growth if the urine is still supersaturated. 4) Meanwhile, non-dialyzable material in the urine has adsorbed upon the crystalline surfaces to make a matrix. 5) And during all this time, partial fusion of spherulites is occurring with development of crystal bridges and other actions that might be described as reworking of the polycrystalline mass. SUMMARY
An attempt is made to apply crystallization phenomena to the pathogenesis of stones and a theory is presented in which stones are looked upon as the result of crystallization processes alone rather than the consequence of some kind of vital activity on the part of stone matrix. The complexity of crystallization processes, especially when dealing with polycrystalline masses, is illustrated with a variety of in vitro and in vivo observations. A tentative schematic formulation of the stone process in terms of crystallization is also offered. The authors believe that the persistent obscurity surrounding the basic mechanism of stone production largely vanishes when viewed in this way.
TnE JorRNAL OF UROLOGY
Copyright© 1966 by The Williams & Wilkins Co.
EXPERIMENTAL OBSERVATIONS ON THE PATHOGENESIS OF URINARY CALCULI C. W. VERMEULEN, J.E. ELLIS
"ND
TE-CHIN HSU*
From the Department of Surgery, University of Chicago, Chicago, Illinois
Ebstein and Nicolaier, in a classical monograph on experimental urolithiasis published in 1891, arrived at a concept of the pathogenesis of stone formation in which the organic matrix was considered as a sine qua non for the process. 1 They believed that the mechanism for stone growth began by some kind of combination between an albuminous niatrix material and certain dissolved minerals. Thereafter the mineral crystallized out of solution so as to mineralize the matrix ground substance in which they had been dissolved. Subsequently other investigators have held somewhat similar views and it has become widely accepted that the stone matrix occupies an extremely important and vital position in the stone-forming mechanism. 2 ,3 In sharp contrast, recent work in our laboratory has led to an hypothesis in which stone matrix is considered not as a crucial element, but rather as an incidental inclusion in stones. This hypothesis arose as the result of experiments in which an attempt was made to imitate stone formation in vitro using a technique reported in detail elsewhere. 4 • 5 Essentially, the method consists in repeated controlled crystallization of stone minerals in a medium of urine using a system where a wire loop serves as a nidus for minAccepted for publication July 2, 1965. Read at annual meeting of American Urological Association, Inc., New Orleans, Louisiana, May 10-13, 1965.
era! deposition" The system can be modified in various ways so as to yield deposits of most of the stone-forming minerals and also of uric acid and cystine. The deposits, or artificial concretions, often assume many of the structural characteristics of authentic calculi. Surprisingly, they also contain a matrix-like component. Further experiments have shown that the matrix arises by incorporation of non-dialyzable urinary constituents into the developing concretion, for if a medium of urine ultrafiltrate is used in place of whole urine, no such matrix component is present. Hence the concretion matrix, though always present in objects prepared in whole urine, is not a necessary constituent. Instead it might be considered as an incidental inclusion that results from surface adsorption of protein upon the crystallizing mineral, a phenomenon well-known in crystal theory. If these in vitro results are extrapolated to the formation of true stones, attempts to understand the pathogenesis of calculi would focus not upon some cryptic vital matrix activity but rather upon crystallization per se. The purpose of the present paper is to elaborate upon this view and, with the aid of further in vitro observations, to develop a general hypothesis of the stone process in terms of crystallization. Some illustrative in vivo experiments are also included.
This investigation was supported by grant AM 475 from the National Institute of Arthritis and
Metabolic Diseases, National Institutes of Health. * Present address: Department of Urology, National Taiwan University, Taipei, Taiwan. 1 Ebstein, W. and Nicolaier, A.: Experimentelle Erzeugung von Harnsteinen. Wiesbaden: J. F. Bergmann, 1891. 2 Boyce, W. H., and King, J. S., Jr.: Crystalmatrix interrelations in calculi. J. Urol., 81: 351, 1959. 3 Cornelius, C. E.: Studies on ovine urinary biocolloids and phosphatic calculosis. Ann. N.Y. Acad Sci., 104: 638, 1963. 4 Vermeulen, C. W., Lyon, E. S. and Gill, W. B.: Artificial urinary concretions. Invest. U rol., 1:
370, 1964. 5 Fried, F. A. and Vermeulen, C. W.: Artificial uric acid concretions and observations on uric acid solubility and supersaturation. Invest. Urol., 2: 131, 1964.
681
IN VITRO EXPERIMENTS
If we are to explain stone formation by means of crystallization phenomena, it will be helpful first to consider certain fundamentals of crystallization, in particular the concepts of supersaturation and nucleation. Both of these terms are occasionally used in an imprecise n1anner in discussion of stone mechanisms. Supersaturation. It is well known that the stone minerals are generally more soluble in urine than in water even at an equivalent pH. JVIany other urinary components-both specific (e.g. citrate) and non-specific-enhance the solubilizing power of urine. However, the supersolubility in urine
682
VERMEULEN, ELLIS AND HSU
SUPERSATURATED
SATURATED
n
acidify ~
D
Uric a. sol 'n
LJ
~
further acidif.
.
spontaneous nucleation
. .
extraneous
nucleation
evap,
t
D
seed with crystal
SUPERSATURATED
______,.
~
primitive ~
polycryst. mass
re-nucleation at crystal
FIG. 1. Schematic illustration of supersatura-
tion and nucleation using uric acid as solute and water as solvent. See text for details.
compared to water is not to be confused with supersaturation. In true supersaturation the amount of dissolved solute is actually greater than is present in a saturated solution in the identical solvent, a condition that can be induced only in special ways. Figure 1 illu,strates 2 methods, both starting from a solution already saturated. In one, the rnlvent power of the medium is slightly reduced by adding acid to decrease the solubility of uric acid; in the other, the nature of the solvent it,elf i, unchanged, but its volume is somewhat reduced by evaporation. If either of these operations is carefully performed and not too drastic in extent, precipitation may be much delayed or fail to occur entirely, as if the solute were reluctant to enter the solid state. Meanwhile, the solution obviously becomes truly super,mturated. It fhould be noted that both methods have their counterparts in the normal processes of urine forrn.ation, for a very dilute glomerular filtrate is gradually concentrated by reabsorption of water and at the same time modified in a variety of other ways. Therefore, it would be reasonable to find that some of the poorly soluble materials ultimately reach a supersaturated condition and such, indeed, is often the case. Nucleation. The state of supersaturation is clearly an unstable condition and when it becomes excessive, precipitation will eventually develop. But prior to actual precipitation, the phenomenon of nucleation is necessary. By nucleation is meant the appearance within the solution of sub-microscopic aggregates of molecules (uric acid molecules in the illustration of fig. 1) where the aggregates satisfy 2 criteria:
FIG. 2. Development of adherent polycrystalline mass in supersaturated solution of copper sulfate in water. A, original seed crystal of copper sulfate. B, crystal has grown but new crystals have also appeared. C, successive generations of crystals have led to large mass. (Measuring bar in C represents 1 cm. But A and B are magnified 5 times greater than C.)
1) the molecular collection, though formed by random molecular movements, must happen to have its molecular unit, arranged in the proper crystal lattice ori2ntation for the particular substance and 2) the,e randomly formed aggregates mmt be of a certain critical size, for if too small they immediately disperse and dissolve back into true solution. Only when aggregates appear which are larger than the critical size will they succeed in growing by acting like miniature seed crystals. Thus, the reason why a supersaturated solution may temporarily remain in a state of apparent stability is that nuclei, as so defined, have not appeared. But as soon a3 nuclei of the critical size are present, they grow rapidly and produce a visible precipitate until, eventually, the state of supersaturation is relieved. Naturally, nucleation becomes more likely as the degree of supersaturation increase3; and if nucleation occurs simply became supersaturation is extreme, it may be designated as of the spontaneous type. However, the degree of supersaturation is not the sole determinant; mere agitation, a scratch on the inside of a glass container, dust particle,, debris, and foreign surfaces may induce nucleation that might otherwise not appear spontaneously. This type of nucleation might be termed extraneom. Additionally, figure 1 shows a special circumstance where new nucleation sites formed at or near the surface of a pre-existent crystal. Though these new sites are often swallowed up by the larger crystal, it is possible for them. to develop into new crystals
PATHOGENESIS OF URINARY CALCULI
683
FIG. 3. Photomicrographs show production of cystine polycrystalline mass in supersaturnted water solution, beginning with single crystal (A) attached to fine glass filament. D (at much lower magnification) shows not only further growth but also reworking, with crystals fusing toward glassy mass. (Magnifications: Measuring bar in D represents 1 mm. But relative to D, A is magnified 40X, B, 30X and C, 20X.)
FIG. 4. Habit modification in artificial concretions of calcium phosphate. A, formed in medium of water, yielding loose powdery deposit. B, formed in urea-salt solution, showing hard concretion with leathery surface. (Measuring bar represents 1 mm.)
684
VERMEULEN, ELLIS AND HSU
FIG. 5. Habit modification of calcium phosphate concretions. A, concretions of adherent spherules formed in medium of urea-salt solution. B, same medium except that, it also contained 10 per cent mg. Concretion is hard smooth crust. (Measuring bar represents 1 mm.)
now attached to the old. In consequence, a polycrystalline mass results, held together by crystal forces alone. Production of simple polycrystalline masses. Figures 2 and 3 illustrate crystal masses formed with substances which tend to produce crystals sufficiently large for easy viewing. In one instance a crystal of copper sulfate was suspended in a supersaturated water solution (fig. 2). At first the crystal simply increased in size but with slight fluctuations in the degree of supersaturation, new crystals soon appeared on its surface. Successive generations followed and eventually a large hard adherent polycrystalline mass developed. Similarly, though at a micro level, figure 3 shows the production of a polycrystalline mass of cystine. The cystine illustration also dern.onstrates, though imperfectly, another phenomenon which might be called "reworking" of the polycrystalline mass. After photographing the object as seen in figure 3,C it was repeatedly subjected to alternating periods of slight undersaturation and supersaturation. As a result, the individual crystals are being reworked, fusing together and producing larger masses with indistinct outlines (fig. 3,D). If reworking were carried to con1pletion, the whole object could become an irregular glassy deposit somewhat like ice.
Artificial concretions and habit modification. The technique previously mentioned for producing artificial urinary concretions can be used with media of simple water solutions as well as with urine. The simpler medium then permits more exact control of its composition and allows demonstration of the remarkable variation in the appearance of the concretion that may result from seemingly insignificant alterations in circumstances, a phenomenon that might be termed habit modification. The concretions shown in figures 4 and 5 are all of calcium phosphate (apatite-brushite composition) but they vary greatly in external appearance and texture. In figure 4,A the medium consisted of plain water and the deposit appeared as a loose fluffy material. However, if the water medium contained urea and NaCl (in concentrations approximating normal urine) a hard leathery object was produced quite different from the previous specimen (fig. 4,B). The object shown in figure 5,A was made in the same urea-salt medium but the ratio of Ca: P was altered somewhat and the concretion habit was changed, now appearing as a mass of fusing spherules. Finally, the effect of adding Mg ion (10 mg. per 100 rn.l.) to the urea-salt medium is shown in figure 5,B; here the deposit has taken on the forn1 of a very hard
685
PATHOGENESIS OF UhlNARY CALCULI
EFFECT OF "MATRIX" ON SPEED OF DISSOLUTION (Solvent O. 05N HCI) WITHOUT "MATRIX"
/WITH
"MA.TRIX"
20-
. Frn .. 6. Calcium phosphate concretions after d1ssolvmg away calcium phosphate. A, objects formed m 10 davs m urea-salt medium with chloroform as a11tibacterial agent. B, objects formed m 5 days aseptically in same medium. In both A and B bare wires (above) are obtained after dissolution. But when albumin, 100 mg. per cent, was added to the medium, matrix residues (below) are found. (Measuring bars represent 1 mm.)
crust. Similar experiments, both with calcium phosphate and other stone minerals, have repeatedly demonstrated the same extreme sensitivity to seemingly minor variations in either the medium or in technical details. Obviously the habit of polycrystalline masses is subject to pronounced alterations dependent upon the environment in which they develop. The condition seems to be somewhat analogous to habit modification of single crystals due to impurities in the crystallizing media, a condition of great interest to crystallographers in recent years. Protein adsorption and matrix. The occurrence of matrix-like components in artificial concretions produced in urine has already been mentioned. Similarly, a matrix is present in concretions developed in simple watery media containing a small amount of protein (fig. 6,A). However, previous publications have pointed out a possible objection to considering such matrices as really analogous to the matrix of true stones, for they could be merely factitious, the result of the bacteriostatic agents necessarily used in preparing the concretions. Ordinarily, either formaldehyde (0.2 gm. per 100 ml.) or a layer of chloroform beneath the urine or water medium has been used in the concretion technique in order to prevent bacterial growth. It is conceivable that the concretion matrix is the result of action by the antibacterial agent in denaturing and precipitating the protein. If so, the supposition that stone matrix arises simply by surface adsorption of unmodified protein would be illusory. Therefore,
HOURS
Frn. 7. Comparison of dissolution speeds of 2 calcium phosphate concretions that were identical in_ weight (58 mg.) and texture, one with and one without an albumin matrix. Concretions suspended and rotated in 60 ml. of 0.05 N HCl as solvent. Increasing concentrations of calcium reflect speed of dissolution. Retarding action of "matrix" is shown.
the experiment illustrated in figure 6,B is significant in resolving the doubt, for here sterility was maintained without using any antibacterial agent, and though the experiment was only of 5 days' duration, a definite matrix was obtained. Thus the matrix-like residue cannot be considered as a simple artifact. Impedence to dissolution by matrix. When concretions with and without matrix are subjected to solvents, it has been observed repeatedly that those with a matrix tend to dissolve more slowly. Figure 7 illustrates the result of one such experiment in which calcium phosphate concretions, identical in weight and indistinguishable in structure, were suspended and continuously rotated in a solvent consisting of 0.05 N HCl. As dissolution progressed, the calcium content of the solvent increased dependent upon the speed of dissolution. It is evident that the concretion without a matrix dissolved considerably more rapidly. Repeatedly, similar tests with other concretions have yielded similar findings. Perhaps an important implication can be derived from these observations if the stone-forming reaction is really a reversible one. In that case a stone would begin to dissolve if the urine concentration fell below the level of saturation. However, the matrix would remain behind to form a barrier to rapid penetration of the unsaturated urine. As a result, dissolution ·would be retarded.
686
VERMEULEN, ELLIS AND HSU
_FIG. 8. A and Bi views of oxamide spherulites formed in urine as seen through polarizing microscope without (A). and with (B) crossed polars. ~OX They consist of spherical clusters of minute elongate cry~tals radially ar~anged .. Can~ D, photomicrograph~ (not polarizing microscope) of oxamide crystallizat10n to show _habit modific~t1on. _Fro1!1 water solut10n (C), blade and dendritic forms appear. When the_ ,ya~er sol~t1on also contams uric acid (5 mg. per cent), oxamide crystallization (D) becomes spheruhtic m habit. 40X. IN VIVO OBSERVATIONS
The purpose in this section is to illustrate in rats rnme of the crystallization phenomena previously described. To do so, an old technique for producing stones in animals was chosen in which oxamide (the diamide of oxalic acid) is administered in the food. The oxamide is excreted as such in the urine. It is a relatively insoluble substance and if given in sufficient amounts, the animals have a severe form of lithiasis. As shown originally by Ebstein and Nicolaier these stones are composed of oxamide and alrn contain a typical matri.'i:.i Indeed, the conclusions by these early workers on oxamide stones are important contributions to the concept that matrix is a necessity in the stone-forming process. Oxamide crystallization. When oxamide crystallizes from urine in either in vivo or in vitro experiments, it assumes a spherulitic form, an interesting type of crystallization seen with many substances, including some of those present in human calculi. When the oxamide spherulites
were examined with the polarizing microscope many elongate crystals all radiating from the center of the sphere were noted (fig. 8,A and B). Oxamide characteristically crystallizes from both urine and urine ultrafiltrates in the spherulitic form but from water it appears as small blades which may sometimes repeatedly branch to assume a dendritic form (fig. 8,C). However, spherulites are not invariable in urine, for if crystallization is sufficiently rapid, the blade and dendritic form appears. Contrariwise, spherulitic crystallization can occur from water solutions if certain impurities are also present. Figure 8,D shows the effect of an impurity consisting of a small amount of uric acid (5 mg. per 100 ml.) present in the crystallizing medium. Here, then, is an illustration of habit modification in oxamide crystallization. The early stone lesion. When oxamide is given to rats in high dosage (1.2 gm. per 100 gm. of diet) for 2 or 3 days, excessive supersaturation results in nucleation. In consequence, spherulites
PATHOGENESIS OF URINARY CALCULI
. FIG. 9. E_arly lesions of renal papillae in rats given oxamide. A, small spherulites have formed and are lined up in lumens of collecting ducts. B, stone formmg upon papillary tip exhibiting spheruhtic growth. Below stone has been teased from papilla. On its inner' surface finger-like proJ ect10ns are seen where stone extended into dilated ducts. (Measuring bars represent 1 mm.)
appear in the urine. Immediate examination of the fresh kidney with the dissecting microscope may reveal an interesting early lesion in the renal papilla. Figure 9,A shows large spherulites lined up in the lumens of some of the papillary ducts. A somewhat later le~ion is seen in figure 9,B where a minute stone has developed upon the papillary tip. vVhen the stone was teased from the papilla, its inner aspect showed projections which previously extended up into the ducts. One gets the impression that some of the larger spherulites were unable to pass through the openings of the ducts at the papillary tip and that the retained spherulites then acted as growth centers for development of a papillary stone or encrustation. If rats are maintained on this high dosage, death will occur within 14 days, but at autopsy papillary lesions are usually no longer found. Instead, calculi are present throughout the urinary system. Evidently, the initial papillary stones sloughed away from the papilla; and while most of the fragments were no doubt discharged with the urine, others were retained in various parts of the urinary system to act as further growth centers. The resultant stones still show evidence of spherulitic crystallization (fig. 10) but as new crystals are laid clown upon them, the spherulites grow and fuse into a unified mass. Experiment on supersaturation with and without nucleation. It has previously been shown that if oxamicle is given in an amount which, of itself, is insufficient to produce stones spontaneously, a
687
FIG. _10. Surface_ of oxamide stone. Spherulites of varymg size fusmg mto unified mass. (Measuring scale is in mm.)
foreign body implanted in the bladder will nevertheless result in oxamicle stone formation throughout the urinary system. 6 The experiment was interpreted as showing that the foreign body acted by inducing nucleation (extraneous type) when the urine was supersaturated, but not to the degree where nucleation would occur spontaneously. In the experiment reported in the present paper, nucleation is provoked in another way~ by initial administration of a shock nucleating close of oxamicle. Thus the animals receive no operation or surgical trauma to produce a local lesion. Instead, a preliminary high close of oxamide is used simply to initiate nucleation. Thereafter, treatment is continued with the lower dosage. In this way it might be possible to shavv the significance of nucleation as an essential factor in stone production without any operative insult to the urinary system whatever. Four groups of adult Holtzman female rats were used. One group, the primary control, received 0.3 gm. oxamicle per 100 gm. of Purina laboratory chow diet throughout the 4-week experiment. Another group was given the same dosage except that a nucleating shock close (1.2 gm. per 100 gm.) was used for the first 3 clays. Two secondary control groups were also included: in one of them, the shock dose was given for 4 clays and the 21 animals were sacrificed immediately thereafter and, in the other, the shock close was also given for 4 days but oxamicle was then completely 6 Gill, W. B. and Vermeulen, C. W.: Oxamide crystalluria and urolithiasis, rat and in vitro observations. Invest. Urol., 1: 339, 1964,
688
VEI::WEUL:EN, ELLIS AKD HSU
Fm. 11. Oxamide stones formed in control rats on low dose. Each white bar indicates 1 rat. Stones were found in only 1 animal; they are shown in their approximate original anatomical locations. White bars are 15 mm. long.
Fm. 12.0xamide stones formed in rats where high shock dose was used initially to induce nucleation followed by low dose used in control group. All rats showed stones distributed in various parts of urinary system.
discontinued. These 22 animals were sacrificed at 4 weeks. Comparison of stone growth. Of 20 rats in the primary control group (where the initial nucleating dose was not used) stones were found in only 1 rat at the end of the 4-week experiment (fig. 11). However, all 21 rats of the experimental group showed stones widely distributed throughout the urinary system (fig. 12). It is obvious that the initial heavy dosage had a profound effect. But were these stones already completely formed during the first 3 days, or did they continue to grow in the period on the lower dose? One of the secondary control groups (shock dose for 4 days, then sacrifice) provides an answer, for only the expected minute papillary lesions,
along with fine spherulitic sand, could be seen when the urinary systems were examined with the hand lens. Stones such as those shown in figure 12 were not observed. Clearly, then, marked stone growth continued during the 25 days when the rats received the low dose. The experiment illustrates that stone initiation requires both supersaturation and nucleation. The nuclei then grow into crystals or minute polycrystalline masses (spherulites when oxamide is used) and if, by chance, any of these embryonic stones are retained in the urinary system, they have an opportunity to grow-provided the urine is still supersaturated. However, if the urine is sufficiently undersaturated, the embryonic stones will dissolve or be discharged with the urine, as
PATHOGENESIS OF URINARY CALCULI concentration etc.
A
spontaneous
\
- - - - - - nuclearion
ISUPERSATURATION I
extraneous
~
I
/
NUCLEATION
I CRYSTALLIZATION I/
t
.1
\
nucleation
/
cc:1::tion
POLYCRYSTALLINE/ MASS habit modifiers
/
t
'
"reworking"
adsorbed ''matrix"
B
no "matrix"
REVERSIBILITY AND DISSOLUTION WHEN UNDERSATURATED
DISSOLVED STATE
-----'------"'
~
~
SOLID STATE
~
\ "matrix" adsorbate impedes dissolution
FIG. 13. A, schematic formulation of stone producti?n viewed as crystallization process. B, expression of possible significance of matrix in retarding dissolution. I
i i
"' , I
shown in the other secondary control group. Here (where only the shock dose was given followed by 24 days without oxamide) all the rats were completely free of stones or papillary tip lesions. DISCUSSION
It is self evident that stone production must be intimately linked with phenomena concerned with the insolubility of certain substances in urine. But a stone is not a mere precipitate. Stones have an organized wholeness, are sometimes bizarre in external structure (e.g. jack stones) and often have a complex internal architecture (with lamination, radial striation, etc.). It would seem, then, that insolubility and factors controlling it would be insufficient to explain such a complex thing as a stone, though perhaps adequate to account for crystalluria. Something more than mere precipitation seems to be required to produce a stone. And "something more" is available, for all stones have an organic matrix. In consequence, matrix is commonly thought of as a cement holding the mineral together, or as a skeleton accounting for the complexity of stone structure, or, again, as a ground substance into which the mineral is deposited under the guidance of the matrix. These concepts seem to offer a bridge over the gap between precipitation processes on the one hand, and stone on the other. In this paper, on the contrary, stone matrix is given no such vital role. Instead, matrix is considered as an incidental inclusion, a notion arising
689
primarily fron, previous work on artificial concretions. If true, then the role of matrix as a bridge between simple precipitation and the complex wholeness of stones is lost, and it becomes necessary to find another explanation to take its place. Perhaps crystallization itself can cover the conceptual gap when it is realized that the field of crystallization concerns far more than the production of a mere precipitate or the development of single crystals. An amateur excursion in the field appears to indicate that the special roles ordinarily given to matrix can be accounted for by crystallization processes themselves. The miscellaneous experiments and observations presented in this paper are intended to suggest how a theory of basic stone-forming processes can be built around conditions affecting crystallization by considering a stone as a polycrystalline mass. Sometimes the mass is composed of very large crystals, as in calcium oxalate dihydrate or cystine stones, but usually the crystals are very much smaller and more or less fused into a mass not obviously crystalline in nature. Schematically, some of the principal factors concerned in crystallization are given in figure 13, a formulation which may serve to correlate the various experiments that have been presented. In the section on in vitro experiments, some of the basic principles of supersaturation and nucleation were discussed in some detail. Simple illustrations of polycrystalline masses were also given to indicate that a matrix cement is quite unnecessary to hold the crystals together (as is also illustrated, of course, in ordinary rocks and minerals). Evidence was also presented to supplement previous reports showing that the existence of matrix could be accounted for by surface adsorption of non-dialyzable material that is invariably present in urine. Further, some in vitro studies suggest how the complexity of structure and the peculiar, seemingly non-crystalline, texture of stone material could be explained by reworking and habit modification of the polycrystalline mass. Lastly, an experiment was reported to show that a matrix of adsorbed protein, though it does not govern the laying down of mineral, still can perform a function by impeding dissolution ,vhen conditions for demineralization might otherwise be suitable. The experiments with oxamide are in vivo illustrations of this attitude toward stone forma-
690
VERMEULEN, ELLIS AND HSU
tion as a crystallization process. The pronounced tendency toward spherulitic crystallization is helpful here not only as a means of identifying the early stone lesions, but also as an example of habit modification. Further, reworking and fusion of the primary crystalline units are illustrated upon examining the developing stones. The necessity for nucleation, in addition to supersaturation, could also be demonstrated by using the technique of an initial shock dose followed by a prolonged period on a dosage that is insufficient, of itself, to produce calculi. We might therefore reconstruct the course of stone formation here as follows: 1) Oxamide, a relatively insoluble substance, leads to supersaturation; and, if supersaturation is sufficiently pronounced, results in spontaneous nucleation. Oxamide crystallization ensues, occurring as spherulites because of habit modifiers as yet unidentified. (Though the in vitro experiments show that uric acid is such a modifier, it must be remembered that uric acid does not occur in rat urine in very significant amounts.) 2) Most of the spherulites are discharged with the urine but some of these forming in the papillary ducts fail to pass through the papillary ostia. These act ar foci for further crystallization, again in the spherulitic form. 3) After the papillary lesions slough off, most of
the fragments are probably expelled with the urine but some remain behind, either because of their size or by pure chance, and act as giant nuclei for further growth if the urine is still supersaturated. 4) Meanwhile, non-dialyzable material in the urine has adsorbed upon the crystalline surfaces to make a matrix. 5) And during all this time, partial fusion of spherulites is occurring with development of crystal bridges and other actions that might be described as reworking of the polycrystalline mass. SUMMARY
An attempt is made to apply crystallization phenomena to the pathogenesis of stones and a theory is presented in which stones are looked upon as the result of crystallization processes alone rather than the consequence of some kind of vital activity on the part of stone matrix. The complexity of crystallization processes, especially when dealing with polycrystalline masses, is illustrated with a variety of in vitro and in vivo observations. A tentative schematic formulation of the stone process in terms of crystallization is also offered. The authors believe that the persistent obscurity surrounding the basic mechanism of stone production largely vanishes when viewed in this way.