Exp. Path., Bd. 10, S. 1-27 (1975)

Department of Pathology (Direetor: Prof. Dr. Bo THoRELL) Karolinska Sjukhuset, Stockholm, Sweden and Department of Pathology, University of Odense, Odense, Denmark

Urolithiasis induced with DL-3,a-dimethyltyrosine methylester Hel I. A scanning electron microscopic investigation

By S. A. G. DATSIS

With 43 figures

(Received January 31, 1974)

Key-words: urinary traet; kidney; bladder; urolithiasis, renal injury; DL-3,a-dimethjltyrosine methylester HCL; sranning electron microsropy; rat

Summary

Scanning electron microscopic investigations on rats subjected to DL-3,a-dimethyltyrosine me­thylester HCl treatment, indicated that the drug in reference induces renallesions substantiated by proximal tubular epithelial cell regreSsion and eventual exfoliation, coupled by formation of 3,a-dimethyltyrosine calculi both in the upper and lower segments of the urinary tract. The develop­ment of calculi was found to be preceded by a moderate to massive precipitation of a fibrin-like substance intermixed with blood cells, and with an occasionally concomitant deposition of exfoliated cells of epithelial origin, to be succeeded by precipitation of crystals.

Stone in the urinary tract is one of the oldest disease entities from which the human race is said to have suffered. It can be taken as axiomatic that renal stones are composed of substances conveyed to the kidney and subsequently deposited.

In 1937, RANDALL made a masterly review of all current theories of stone formation; entirely by a process of deduction, he decided that there must be apre-existent lesion in the renal pelvis to which a calculous eould remain anchored during the period of growth. He fQund the lesion in the renal papilla, currently known as "Randall's plaque", and postu­latedthat this was the underlying cause of renal calculus. On microscopic examination, he cOllsidered these plaques to represent plaques of calcium deposited in the interstitial tissue of the renal papilla and proclaimed definitely that the deposits were not intratubular.

ROSE:-;o\\" (1940) and VERMOOTEN (1941) confirmed RANDALL'S findings, though the latter pointed out that papillary lesions may occur independent of any subsequent development of stones. R\NDALL'S theory is currently by and large generally held, but it is very doubtful whether all stones are in fact formed in this manner. It is widely suggested, and has been fairly eonvineingly demonstrated by BUTT et al. (1952), that crystalline constituents of the urine separate out of solution due to a shortage of "protective colloids", and it is a fairly weIl established contention that crystaIline deposits occur as a result of excessive excretion or an abnormal pH of the urine.

ANDERSON (1946) studied the small crystalline deposits on microscopic calculi in the kidney substance; he defined a microscopic calculus as a deposit of calcareous material in the substance of the kidney tissue proper, of a size sufficient to be examined easily under the ordinary low power microscope lens, and with a diameter ranging between five or six times that of anormal tubular epithelial cel!.

The aim of the present investigation was to construct an experimental model facilitating the elarification of previously unresolved or thoroughly controversial hypotheses pertaining

1 Exp. Path. Bd. 10, H. 1/2 1

urolithiasis, utilizing DL-3,a-dimethyl-tyrosine methylester HCI, a specific competitive in­hibitor of tyrosine hydroxylase.

a-methyl-tyrosine (a-MT) has been repeatedly shown to have the capacity of inhibiting tyrosine hydroxylase (SPECTOR et al. 1965, WEISSMAN and KOE 1965, MAITRE 1965, MOORE 1966). The methylester of a-methyltyrosine possesses comparable properties by virtue of a rapid hydrolysis in vivo to the eorresponding amino acid. a-methyltyrosine is hydroxylated to a-methyldopa (MAITRE 1965, UDENFRIEND et al. 1966, REICH et al. 1966), which is in turn decarboxylated to a-methyldopamille, the latter amines being known to act as false transmitters. In view of this potential, the use of a-methyltyrosine has been widely adopted as me ans for the study of the sympathetic nervous system. However, MOORE et al. (1967), have illdicated that the interpretation of the data obtained in some investigations utilizing a-methyltyrosine might be complicated by the toxic effects of the drug in reference. a­methyltyro~ine has been ShOWll to induce renal damage in the rat, and to produce crystal­luria coupled with urolithiasis in the dog (ENGELMAN et al. 1968). The pathogenesis of the renallesion in the rat remains by and large a speculative hypothesis and a matter of contro­versy.

The related compound DL-3,a-dimethyltyrosine methylester HCl (H 59/64) also acts as a potent inhibitor of tyrosille hydroxylase (HANsoN 1967). This compound has been shown to induce renallesions with formation of urinary calculi in mice (MAGNUSSON et al. 1968).

Materials and methods

Throughout this investigation, adult, male Sprague-Dawley rats, ranging in weight from 180 to 200 g were used. The animals were given food and drin king water a d li bit u m.

For the aim of the present investigation, the gastric-tube mode of administration of DL-3,a­dimethyltyrosine methyltyrosine methylester HCl appeared to be most suggestive. Preliminary ex­perimental trials indicated that intraperitoneal administration of the drug resulted in a considerably increased number of fatalities among the experimental subjects. On the other hand, 3,a-dimethyl­tyrosine methylester HCl mixed in the food in powder form resulted in a remarkably slow and un­even development of the lesion. This latter proeess would not allow for a possible calculation of the dose which manifests thc symptoms of the sublethai toxicity, much less would it reveal the primary steps in the formation and growth of ealculous.

DL-3,a-dimethyltyrosine methylester HCl, obtained from the Sigma Chemieal Co., (St. Louis, Mo. U. S. A.), was dissolved at a dose of 600 mgjkg of body weight, in 10 ml of distilled water per experimental anima!. A placebo group was granted preference over an ordinary control group, as it could by no means be excluded that the stress connected with the gastric intubation might in­fluence the animals' overall "thriving".

Groups of 5 experimental and 3 placebo (control) subjects were sacrificed at 15 minute intervals following a single administration of 600 mgjkg of body weight of 3,a-dimethyltyrosine methylester HCI. The animals were anesthetized by an intraperitoneal injection of ~embutal (Sodium Pento­barbital) at a dose of 35 mgjkg of body weight. The dose was controlled so that the animals became insensitive to painful stimuli without passing through an exeitatory stage. The abdomen was opened by means of a ventral midline ineision, from the level of the pubis to approximately the level of the diaphragm; the intestinal mass was gently displaeed, the renal stalks clamped with hemostats and the kidneys, ureters and urinary bladders were rapidly removed.

In order to avoid papillary injury, the upper and lower poles of the kidney were first dissected away by transverse sections passing through the pelvis above and below the papilla. The specimen was laid open by a cut on its medial aspect, applying fine surgical scissors and avoiding any contact with the papilla. It was then immersed in 6.25 % glutaraldehyde for 12 hours. (The commereially available 25 % glutaraldehyde was filtered twice through activated charcoal and buffered with 0.1 M phosphate buffer to pH 7.35). Following fixation, the specimens were postfixed in 0.1 M phos­phate buffered 1 % OsO. for six hours, freeze-dried under absolute vacuum and were double gold coated prior to examination under a Cambridge S4 scanning electron microscope.

The urinary bladders were processed through the same channels prior to examination under the scanning electron microscope.

Identification of DL-3,a-dimethyltyrosine in the urinary calculi was performed as folIows: the caleuli were dissolved in the least possible amount of 1 N HCl. The amino acid was precipitated by saturated sodium acetate solution, filtered, washed with water and dried. Chemieal identifieation was done by means of the IR-speetrum (in KBr) which was notably indentieal with t~at of t~e authentie 3,a-dimethyltyrosine, and by X- Ray crystallography. The methodology apphed for m X-Ray crystallography will be published elsewhere (DATSIS 1974a).

2

Fig. 1. Low magnification scanning electron micrograph illustrating a glomerulus from a rat at 15 minutes following the administration of DL-3,a-dimethyltyrosine methylester Hel. No signs of alterations of a pathologieal nature are discernible. X 540.

Fig. 2. The Bowman's capsule partly enclosing the glomerulus in fig. 1, as weil as the glomerulus itself exhibit an apparently normal architectural plan. The epithelial-cell foot processes (podocytes) displayaregular orientation devoid of evidence of fusion and/or swelling. X 5,400.

1* 3

Fig. 3. High magnification micrograph of apart of the glomerulus depicted in fig. 2. The normal interdigitation of the epithelial cell foot processes is clearly displayed. X 5,040.

Fig. 4. Survey scanning electron micrograph illustrating portion of the luminal surface of the col­lecting tubule depicted in fig.1 (adjacent to the glomerulus). The free surfaces of the epithelial cells, slightly buldging into the tubular lumen, display signs of a moderate deposition of a fibrin­like material. Note that these cells are devoid of a brush border. X 1,920.

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Fig. 5. Orienbtion scanning electron micrograph of a section of the renal cortex from an experi mental subject at 17 minutes. Three proximal convoluted tubuli, recognizable by virtue of the pre sence of a brush boder, are seen in the foreground. In the upper portion of the micrograph, there is a segment of a collecting tnbule at longitndinal section. x 480.

Results

A. Upper urinary tract (kidney)

Irrespective of the duration of the present experimental study, no discernible pathological in nature alterations were present in the renal glomeruli of the laboratory subjects subjected to 3,a-dimethyltyrosine methylester HCI treatment, at least at the scanning electron miero­seopie level, with the Bowman's capsules and epithelial eell foot proeesses (podoeytes) in an order not notably different from the normal anatomical morphology (figs. 1-3).

The earliest diversion from the normal appearanee was depieted in the eortieal portions of the collecting tubules, substantiated by progressively inereasing deposits of a fibrin-like material, intermixed with oceasional blood eellular elements, situated On the luminal surfaee of the tubular epithelial eells (figs. 4-7). By far, the most profound toxie effeets on the renal eomponents were evident in the proximal convoluted tubules at 16 minutes following the administration of a single dose of 3,a-dimethyltyrosine methylester HCI. The epithelial eell brush border exhibited fairly weil eireumscribed regional damage, coupled by alterations in the epithelial eells, the latter displaying signs of cellnlar death, ranging from simply in­ereased porosity of the cellular membrane, to final exfoliation. A eonsiderablc number of exfoliated epithelial eells, occasionally bearing remnants of preserved brush border on them, were easily detectable in the lumina of the proximal eonvoluted tubules (figs. 6, 8-14).

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6

7

8

Fig. 6. Survey scanning micrograph illustrating a section of the lumen of the collecting tubule de­picted in fig. 5. Note the increasing rate of deposition of a fibrin-like substance over the luminal surface of the tubular epithelial cells. About X 1,800.

Fig. 7. High magnification micrograph of a conglomerate of deposited fibrin-like material. The component fibers appear intermingling, thereby forming a net-like structure. About X 1,800.

Fig. 8. Scanning electron micrograph of a portion of a proximal convoluted tubule (cross section) at 17 minutes. A number of epithelial cells appear protruding into the tubular lumen. About X 1800.

Fig. 9. Survey scanning micrograph of a portion of the walls of the tubule depicted in fig. 8. Despite the ill-defined cellular contours, the brush border is apparently weH preserved. About X 4500.

Fig. 10 and 11. High magnification scanning electron micrographs displaying a number of epithelial cells protruding into the lumen of a proximal convoluted tubule; the epithelial ceHs exhibit an ap­parently increased porosity of the cell membrane. Furthermore, the location of these cells may be conceived as designating a process of exfoliation. Portions of the brush border appear to be firmly attached to the epithelial cells. X 9,600; X 19,200.

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Fig. 12. Oblique view of a proximal convoluted tubule (cross section) at 20 minutes. The notably increased number of exfoliated epithelial cells seems to be proportional to the time period that ellap­sed foHowing one application of the drug. Except for fairly weil circumscribed regions of apparent injury, thc epithelial cell brush border displays a rather normal appearance. X 1,600.

Fig. 13. Survey scanning micrograph of the epithelial cell depicted in the center of fig. 12. The pre­sumably exfoliated ceH appears resting on the well-preserved border of intact epithelial ceHs. A pro­found injury to the cell membrane is discernible. X 8,000.

Fig. 14. High magnification micrograph of an epithelial cell from the same specimen. The state of death of this exfoliated cell is elearly indicated by the remarkable injury to the cell membrane. X 8,000.

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Fig. 15. Low magnification scanning micrograph of the papillary tip region of the kidney from an ex­perimental subject at 30 minutes. The openings of the collecting ducts facing the renal pelvis are occluded by a heterogeneous material, presumably obstructing the free f10w of urine. No detectable lessions corresponding to "R,\NDALL'S plaques' are displayed. X 4,800.

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Fig. 16. Survey scanning d ectron micrograph of the opening of one collectin~ duct at the papillary tip. A heterogeneous complex composed of blood cellular elements and exfoliated ceIls of epithelial origin, in a fibrin-like network is seen occluding the ductal orifice. About x 3,730.

Fig. 17. Se::mning electron micrograph iIIustrating the material depicted in fig. 16. With the excep­tion of erythrocytes adefinite identifi cation of its constituents appears rather difficult, if possible at al l. The parietal epithelium of the papilla exhibits an all normal microscopic appearanee. About x 3,750.

Fig. 18. High magnification scanning micrograph of a portion of th e heterogeneous material de­pict ed in figs. 16 and 17. A crystal-fibrin-like complex appears contributing major part to the body of the occluding mass. About X 8,150.

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Fig. 19. A well-defined complex of exfoliated ceHs, blood-cellular elements, crystalline material and a fibrin-like network is seen located on the parietal epithelial surface of the renal papilla. Despite an existing apparent resemblance between this deposit and the "RANDALL'S plaque", no underlying lesion on the epithelium is detee;table. About x 7,650.

Fig. 20. High magnification seanning electron micrograph illustrating the massive deposition of 3,a-dimethyltyrosine crystals in the lumen of a collecting duct. A network of fibers of a fibrin-like appearance can be conceived as determining the spacial orientation of the crystals. x 10,980.

Fig. 21. Scanning micrograph of the lumen of a collecting duet at 30 minutes. A profound preeipita­tion of erystals in a random orientation, together with a fibrin-like network appears obstructing the tubular lumen. X 10,980.

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The rate of epithelial cell exfoliation, witnessed by the number of cells devoid of an apparent connection with a basement membrane, appeared to be directly proportional to the time period ellapsing following a single administration of the drug.

Essentially, a comparable proportionality became evident at the region of the papillary tip, taking into account the amount of heterogeneous material filling the openings of the papillary ducts, and eventually protruding freely into the renal pelvic space (fig. 15). This material consisted by and large of exfoliated rells, blood cellular elements, and crystalline material intermixed with a fibrin-like material in a network formation (Figs. 16-21).

B. Lower urinary tract (bladder)

In the course of the investigation, no detectable deviations from the normal anatomical morphology were present in the urinary bladders of the experimental subjects during the first 15 minutes. The Iimiting Walls of the bladders appeared mostly in a folded arrangement, with clearly defined surface8 and free of any signs of direet or indirect toxic effects. Occasional­Iy occurring erythroeytes could easily be conceived as preparation artefacts (figs.22, 23).

The progressive deposition of organic and crystalline material which eventually resulted in the formation and growth of multiple calculi occurred very rapidly. Fibrin-like material appeared covering and obscuring as a light net the free surfaces of thc walls of the urinary bladders (figs. 26, 27, 29, 30), to become mixed with blood cellular components (fig. 28), thereby forming the matrix of the stone (figs. 31-33).

Heterogeneous in gross appearance masses, which consisted by and large of blood born elements and crystals in a "honeycomb" pattern were literally filling the urinary bladders (figs. 34-40). Though difficult to recognize definite 3,a-dimethyltyrosine crystals at times, the latter were most clearly depieted in fortuitous orientations of the speeimens under examination (fig. 41).

The end-point of the urinary bladder stone formation, taken at the end of 45 minutes, was marked by the formation of large eoneretions, most often intermingling with each other, thus presentillg the false image of a large calculous filling the urinary bladder cavity in its whole (fig. 42).

Discussion

Although it would rather premature to suggest a meehallism for the DL-3,a-dimethyl­tyrosine methylester HCI-induced renal injury, short of a correlative study of the lesion at the submieroseopie level, it might be beneficial to venture the risk of suggesting a scheme whieh eould provide a preliminary basis for further studies.

Results obtained and reported on in the present investigation, profoundly indicate that the epithelial eells of the straight portion of the proximal tubules received the greatest amount of damage. This is the area where the glomerular filtrate loses much of its water content and, perhaps, it is the region where 3,a-dimethyltyrosine is poorly soluble at the hydrogen ion eoneentrations of most body fluids (pH 5-8). The drug, therefore, may exeepd its solu­bility and erystallize in the tubule causing meehanieal damage to the epithelial rells. The protective effects of water loading (MAGNUSSON et al. 1968), provide substantial evidence in support of this speculative hypothesis. It is, on the other hand, possible that 3,a-dimethyl­tyrosine is reabsorbed by the cells in the proximal tubules, exerting its effects during this process. The injury, could also be brought about by tubular seeretion of the drug. Further­morp, 3,a-dimethyltyrosine may cause alterations in the renal blood f1ow, thereby inducing focal tubular ischemia which aggrevates a direct toxic effeet on the tubular cell, resulting in cell necrosis and eventual exfoliation.

Identification of 3,a-dimethyltyrosine as a constituent of the urinary calculi indicated that this eompound is a metabolite whieh is excreted by the kidneys following administration of its methylester.

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Fig. ~2 . Low magnifica,tion scannin g micrograph illustmt ing the urinary bladder mucosa. at 15 mi­Hutes. Crystalline deposits are totally absent. Minor eontaminants could readily be conteived as Ill ere prepluation arte fa ets. X 125.

Fig. ~3 . Snrvey mierograph of a section of the bladder mucosa depictecl in fig. 22. No toxie effects or eonseqllences thereof are detectabl e at this t ime period. X ~50.

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Fig. 24. Low magnification scanning micrograph illustrating the urinary bladder mucosa at 30 minutes. Amorphous deposits obstruct the normal surface. X 16.

Fig. 25. Survey scanning electron micrograph of a portion of the urinary bladder mucosa in fig. 24. Deposits composed primarily of a fibrin-like material are seen obliterating the mucosal surface. No well-defined deposits of a crystalline nature are discernible. X 36.

Fig. 26. High magnification scanning electron micrograph displaying an area of the bladder mucosa. The cellular surface is covered by a network of fibers; blood cellular elements and inorganic material are literally totally absent. X 127.

Fig. 27. Though rather difficult to state with any degree of certainty on the nature of the fibrinous or fibrin-like material extending over the bladder mucosal surface, it is strongly suggestive that the undetermined substances resembles fibrin. X 240.

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.Fig. 28. A moderate deposition of fibrin-like substance, arranged in a network of fibers covering the mucosal cell free surfaces, coupled by the occurrence of blood cellular elements in an intimate relation to the fibers, appear to form the primary stages in the formation of the matrix part of the calculus. X 2,100.

2 Exp. Path. Bd. 10, H. 1/2 17

Fig. 29. Survey scanning electron miuograph illllstrating portion of the bladder mucosa at 35 min­ntes. A den~e coating of a rather homogeneons eonsisteney, with a few well-defined fibers resembling fibrin, extends over the entire mncosal surface, obliterating to a great extent the anatomieal morpho­logy of the mncosa. X 2,340.

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Fig. 30. High magnifieation scanning micrograph of the fibrin-like "stroma" depieted in fig. 29. A remarkably dense network, eomposed of interconneeting fibers, arranged either at random or in regular strands, provide a firm support for cellular elements (upper part of micrograph), presumably the result of a toxiration of the drug on the tubnlar epithelial rells with a subsequent exfoliation andfor a concomitant hemorrhage in the upper nrinary tract (kirlney tissue proper). X 5,850.

2' 19

Figs. 31 and 32. Survey scanning electron micrographs displaying the further development of the mim,ry calculus subsequent to the deposition of a fibrin-like sabstance. The fibrin-like fibers appear to attain and maintain a ragular "honeycomb" arr.1ngement, concurrently becoming intermixed with bloo:! cellular elements, exfoliated ceHs and necrotie tubular epithelial ceHs, and a fairly well­defined crystalline material (38 minutes). X 1,600; X 1,600.

Fig. 33. High magnification of one of the cellular elements depicted in fig. 32. A fibrin-like sub­stance deposited on the cell surface in the form of droplets and/or fibers constitutes the "connecting bridges" between ceIls and crystaIline material, providing a firm framework in spaee for the con­:stituents of the eventual calculus. X 8,000.

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Fig. 34. Scanning electron mierograph of a portion of the urinary bladder cavity at 42 minutes following the administration of thc dmg. Multiple foei of pr :cipitated heterogeneous masses dis­playing points of apparent mutual interconnections are seen at this time intervaL It merits mention­ing at this point that the depicted zones of separation b2twe3n adjacent calculi foundaticms might be apreparation artefact. x 145. Fig. 35. Survey scanning electron micrograph illustrating one of the calculus foei in fig. 34. An ap­preciable number of cellular elements are clearly discernible situated on a "hGneycomb-like" com­plex. x 375. Fig. 36. High magnification scanning micrograph of apart of the heterogeneous mass depicted in fig. 35. The cellular elements mj~ht readily conceived as formin6 the center of balance for the further growth of the calculus. Fibrin-like ami crystalline material are arranged at more or less regular angles with respect to an axis passing through the cello x 1,470. Fig.37. The wide spectmm of variations in the architectural plan of the stone ranged from areas of large cellular popalations to those practically completely devoi:l of cellular elements. The lattu areas appeared to be composed of erystalline and fibri:t-like plaques, arranged at regular geometrical figures. x 1,440.

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Fig. 38. Low magnification scanning electron micrograph depicting a concretion (longitudinal sec­tion) Note the tube-like structures prenmably composed of crystalline material, along with inter­mixed fibrin-like fibers. x 1,470.

Figs. 39 and 40. High magnification micrographs of areas from the calcareous material in fig. 38. Plaques of a crystalline consistency, arranged at right andjor oblique angles with respect to their neighboring counterparts, and with fibrin-like fibers interwining, are the main constituents of the 3,a-dimethyltyrosine calculi. X 3,720; X 3,720.

Fig. 41. Examination of the specimen at an oblique angle reveals the crystalline nature of the formed urinary bladder ealculi. 3,a-dimethyltyrosine crystals closely resembling the authentie 3,a­dimethyltyrosme crystals are depieted been situated on a "honeyeomb-like" network. The apparent continuity of these crydlls with the material compo3ing the supporting foundation, provides eon­vineing evidenee in favor of a crystalline complex. X 1,320.

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Fig. 42. Low magnification scanning electron micrograph of the urinary bladder at 45 minutes. The entire c'avity appears filled at its greatest extent by a calcareous deposit, highly obliterating the margins of the bladder mucosa. X 19.

Amino acid stone formation in the urinary tract is known both from spontaneous and experi . mental conditions. Cystinuria, which occurs as a hereditary dis order in man and dog is characterized by formation of urinary cystine stones (BOSTRÖM and RAMBRAE US 1964, CORNELIUS et al. 1967). The cause oI this fa.milial cystinuria is considered to be a decreased capacity of the rena.] tubule3 to re­absorb the amino acids cystine, lysine, ornithine and arginine (DENT and ROSE 1949). Phenylalanine fed in excess to young rats gives rise to formation of crysta.]s oi phenylalanine and tyrosine in the ureters and bladder (DoLAN and GODIN 1966), a phenomenon bearing resemblance to the experimental model under discussion. A common feature of t hes e amino acids is that they are rather poorly soluble in water at the pR range of the body fluids.

From clinieal and experimental work it is firmly established that crystals appear in the renal tubules following poisoning with sulfonamides and ethylene glycol. Sulfonamide crystals oeeur mainly in the collecting tubules (S~nTH and J ONES 1961). In ethylene glycol intoxications, crystals of cal­cium oxalate are formed. By contrast to the sulfonamide erystals, they are localized both in the proximal renal tl1 bules (J Ö"SSON und RUBARTH 1967) and in the collecting counterparts (DATSIS 1974 b).

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Physico-chemical aspects of urolithiasis with reference to 3,a-dimethyl-tyrosine methylester HOl

The presence of the two main weil established constituents of renal stones, namely, matrix and mineral or crystal, has given rise to 2 general concepts interpreting stone forma­tion. One school considers the first and main step to bc the synthesis of thc matrix, pre­cipitation of the mineral being then a secondary step occurring once the stone is formed (BOYCE and KING 1959, DL'LCE 1958). This view is supported by the fact that there is a e10se morphological relation between the matrix and the inorganic part; it merits mentioning in this connection that following demineralization, the former, indeed, reflects exartly the image of the whole stone (BOYCE et al. 1958). Furthermore, the urinary mucoproteins wh ich are in the majority of clinical cases the main cOl1stituents of the stone matrix have been found to differ in stone formers and in normal people both quantitativoly and qualitatively; thus, in patients larger amounts are excreted in the urine (BOYCE et al. 1954, BOYCE and Sw ANS ON 1955) and their immunological behavior differs (BOYCE et al. 1962). Contrarily, the other school considers the formation of tho crystals to be the principal step, the matrix being just passively precipitated (FJNLAYSON et al. 19\31). Thus, it seoms that the important aspect would be the concentrations of inorganic ions in urine in relation to the solubility of the salts which may precipitate.

Whatever theory is correct, the formation of the crystalline phase, representing over 95 % of the total weight of the stones, is certainly of considerable importance. For the sake of e1arity of this event, provision of some basic facts pertaining the rules governing crystal formation is most appealing.

When a salt is in equilibrium in a liquid phase, the concentration of ions in solution is a constant determined by the solubility of the salt. If this ion concentration is larger than solubility may allow for, crystals will evontually grow; should it, on the other hand be of a lower scale, thoy will dissolve until equilibrium is reached again. The solubility of 3,a-dime­thyltyrosine methylester HCI is approximately 1 g/litcr of water at 20 °C. In comparison with other excreted substances this is not exceptionally low. There are, however, 2 factors which might possibly contribute to the observed formation of calculi: (i) the 3,a-dimethyl­tyrosille methylester might be rapidly and cornpletely absorbed after oral application, or alternatively (ii) the ester is rapidly hydrolyzed to 3,a-dirnethyltyrosine which is rapidly excreted.

The acute toxicity study reportcd herein, has revealed that the crystals were deposited by and large in the collecting tubules to a very great extent. The apparent predilection for this site of precipitation should be related to a "supersaturation" of 3,a-dirnethyltyrosine, due to the progressive concentration of the urine in the tubules. However, before urine is claimed to be "supersaturated" with respect to 3,a dirnethyltyrosine, as weil as other salts, various factors such as ionic strength and pH must be considered.

According to the theory of interionic attraction, the activity of an ion, that is the ability to react chemically, is dirninished by the surrounding ions of opposite charge. This inhibiting influenco increases with the number and the charge of these ions both of which are reflected in the concept of ionic strength; indeed, the ionic strength {l is defined by the formula

{l = 1/2 }; cv2,

where }; = surn, c = concentration of each individual ion and v = valence of each ion. Thu~. the actual activity of a definite ion diminishes when the ionic strength increases; it should. therefore, appear rather clear that in urine the real activity of a certain 3,a-dimethyl­tyrosine or calcium ion, as the case most often appears to be, is lower than the activity in water. This fact sheds light on the eyegrounds of the apparently greater salt solubility in urine (ELLIOT et al. 1959) than in water, which, however, is only an artefact due to the non­application of basic physico-chemical principles.

The pH value has also a great influence on the amount of calcium, magnesium, phosphate, oxalate and urate likely to be present in solution. Thus, alkalinization favours precipitation of calcium phosphate (ELLIOT et al. 1961, NEU MAN and NEU MAN 1958) and magnesium

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ammonium phosphate (ELLIOT et al. 1959, PRIEN 1955), while calcium oxalate (PRIEN 1955), and 3,a-dimethyltyrosine are little, if at all, affected by the pH value.

Urine "supersaturation" may find a number of explanations. In the absence of preformed crystals, a given solution may contain many more ions than the solubility of the salt would permit, without, however, precipitating. This can be partly explained by the fact that the ion concentration required to initiate crystal formation is much higher than the concentra­tion necessary for further growth of already existing crystals. The range between the solu­bility and the level of spontaneous precipitation is referred to as the "metastable range" where the solutions are considered to be "supersaturated" (fig. 43). The state of "super­saturation" can, however, be readily disturbed by the mere introduction of some crystals of the same salt, which induce immediate crystallization. This phenomenon will be referred to in the present report as "heterogeneous nucleation" (fig. 43). Such crystallization can also be produced by crystals of another salt of a similar crystalline lattice. The similarity required for effective heterogeneous nucleation depends by and large on the degree of "super­saturation" , a higher degree of supersaturation calling for lesser similarity.

Hetero-.. 0

"- Spontaneous preci pitatiGl'I "0 geneous c: tU

Supersaturation ., nucleation c: 0

., ""-

GI Solubility 0 ::I

u c: GI 0

0

tU E .. Undersaturation c: GI u c: 0 ()

Fig. 43. Schematic outline of SOIllbility, supersaturation and nucleation.

Recently, also crystalline proteins such as collagen (FLEISCH and NEUMAN 1961, GLIM­CHER et al. 1957, SOLOMONS and NEUMAN 1960, STRATES and NEUMAN 1958) or elastin (TA­VES and NEUMAN 1963) have been shown to induce precipitation of calcium phosphate crystals in otherwise stable, although supersaturated solutions. Such nucleating activity is thought to be due to the binding by these proteins of calcium or phosphate in a sterie order presenting some similarity with the position of these crystals in the apatite lattice (GLIM­eHER 1959, 1960, NEUMAN and NEUMAN 1958). The same speculative hypothesis might also explain the role of fibrin andJor other blood plasma components in the formation and growth of the 3,a-dimethyltyrosine stones.

Literature

ANDERsoN, W. A. D., Renal calcification in infancy and chiIdhood. J. Pediat. 14, 395 (1939). BOSTRÖM, B., and L. HAMBRAEus, Cystinuria in Sweden. Acta med. Scand. Suppt 41 (1964). BOYCE, W. H., F. K. GARVEY and C. M. NORFLEET, Ional binding properties of electrophoretirally

homogeneolls pro teins in urine of normal subjects and in patients with renal calculus disease. J. Uro!. 72, 1019~1031 (1954).

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Author's address: Dr. S. A. DATSIS, cto 121 St. Demetrius Street, Thessaloniki, Greece.

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