The effect of O-Carboxyphenyl-β-D-Gluco-pyranosiduronic acid upon the water dissolution of chemical compounds in urinary calculi
Aronson, Robert B.
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Prien and Walker^39 have treated chronic kidney stone formers with aspirin. They obtained clinical evidence showing no new stone growth and inhibition of the growth of existing stones. The rationale behind such therapy is that aspirin is excreted as a glucuronide conjugate in the urine, and the glucuronide increases dissolution of calcium phosphate stones. Previous laboratory studies^39,10 have shown glucuronides (a concentrate of mixed urinary glucuronides, or o-aminophenol glucuronide) to increase the dissolution of tricalcium phosphate. Tricalcium phosphate is neither a major nor even common component of urinary calculi. The purpose of this investigation is to demonstrate the effect of a glucuronide of salicylates on the dissolution of urinary calculi and their major component compounds. The diffuse pharmacological action of salicylates makes critical, in vitro experiments imperative. The ether glucuronide conjugate of salicylic acid, o-carboxyphenyl-β-D-glucopyranosiduronic acid (o-CPG), a known metabolic product of salicylates found in urine^40 and stones of known composition or synthetic components of stones should be used in these experiments. In the course of this investigation evidence of increased dissolution warranted further experimentation into the possible cause of such effects. Aspirin therapy as initiated by Prien and Walker derived from the qualitative experiments of Neuberg, Mandl and Grauer^33,29,30. In these experiments the authors attempted to either dissolve calcium salts or to inhibit their precipitation by addition of naturally occurring organic and inorganic acids. They showed that 1-menthol-Dglucuronic acid solubilizes both calcium carbonate and calcium phosphate salts, that it functions in this way much better than non conjugated glucuronic acid and that incubation of such a mixture with β-glucuronidase leads to precipitation of the salt. The work of Cessi^10 followed in which the solubilizing effect of a glucuronide was observed during the synthesis of the glucuronide by an in vitro biological system. p^32 in the supernatant fluid resulting from dissolution of a solid phase of tricalcium phosphate containing p^32 (placed in the flask with the liver slices) was measured by counting the activity. The increase of p^32 over a control flask was found to vary directly with the glucuronide concentration. Salicylates have been shown to be excreted as the phenolic glucuronide ^40,25,2. Smith^42 has shown that acetylsalicylic acid is probably hydrolyzed in plasma to form salicylic acid. An increase of urinary glucuronides could then be made to occur by the ingestion of various "glucurongenins" (Teague). Prien and Walker^39 have reported an increase of 200 - 400 per cent over the basal level of urinary glucuronides in man during a 2 gm dose of aspirin / day. Glucuronides are manufactured largely by the liver, kidney and mucosa of the alimentary tract^15,23,24. The biosynthesis requires a coenzyme, uridine diphosphate which is thought to react with glucose to produce uridine diphosphoglucose which then may be oxidized to uridine diphosphoglucuronic acid in the presence of DPN^+45. The uridine diphosphoglucuronic acid will then transfer the glucuronic acid to a proper acceptor such as a phenol. The role of ingested glucuronic acid in this metabolic scheme seems to be negligible. Only a small percentage is recovered in the urine^20. Douglas and King using labeled glucurone found the glucuronic acid in the urine to be labeled in such a way as to support the theory that the glucurone breaks to three-carbon fragments. These fragments probably form a six-carbon precursor of glucuronic acid. Urinary tract calculi have been shown by Prien^37 to contain calcium in 90 per cent of 1000 calculi studied. Thirty-three per cent were pure calcium oxalate, 3.4 per cent pure hydroxy apatite and 34 per cent were mixtures of calcium oxalate and hydroxy apatite. Hydroxy apatite is considered to be the main inorganic component of animal bone. The chemical structure of a unit cell is Ca10(PO4)6(OH)2^34,9,4. This crystal is the most common phosphate crystal found in urinary calculi. It is this component of urinary calculi with which this investigation is mainly concerned. EXPERIMENTAL The first series of experiments were designed to find the effect of o-CPG on the dissolution of a urinary tract stone composed chiefly of hydroxy apatite. The degree of dissolution was followed with phosphate determinations, using the colorometric procedure of Fiske and Subbarow.^21 Since uric acid, salicylic acid, salicyluric acid and o-CPG are all found in the urine in increased amounts following ingestion of salicylates, these compounds were examined for their dissolutive effect. The test flask contained 100 mg of a powdered urinary stone composed mainly of hydroxy apatite, and 0.1 mMoles of the test compound. The mixture was placed in a small dialysis bag along with 4-5 glass beads and 10 ml of buffer. This bag was placed in a 50 ml Erlenmeyer flask containing 30 ml of the buffer and a few glass beads. Several drops of toluene were added to prevent bacterial contamination. The buffer used was Michaelis universal veronal buffer, pH 7.8. This pH was chosen because Prien and Walker^39 had found it to be optimal for dissolution of calcium phosphate in their early experiments. The o-CPG was synthesized in this laboratory using the method of Lunsford and Murphey.^28 The flasks were placed in a shaking water bath at 38°C. and aliquots of the external phase in the flasks were analysed at 5 hours and 10 hours. Duplicate flasks were set up for each substance in question and for the control flask which contained hydroxy apatite only. At the end of 5 hours the o-CPG flask showed an increase in the phosphate concentration of 85 per cent over the control. The other substances showed little or no increase. After 10 hours the o-CPG showed an increase of 36 per cent over the control. We conclude that o-CPG increases the dissolution of urinary tract calculi composed mainly of hydroxy apatite. Furthermore, it is suggested from the decreasing per cent of increased dissolution over time that the o-CPG also hastens the equilibrium of dissolution. The experiment was repeated substituting synthetic hydroxy apatite, prepared in this laboratory, for the powdered stone. Estradiol glucuronide was examined as well as o-CPG. After 5.75 hours both glucuronides increased dissolution; the estradiol glucuronide increased the dissolution by 87 per cent and in this case the o-CPG produced a 555 per cent increase. It can be concluded that o-CPG increases dissolution of hydroxy apatite and that it works better on pure hydroxy apatite than on other forms of calcium and phosphate. Other investigators^39,10 have found an increase of about 40 per cent using calcium phosphate. The first possible explanation for these results, which occurred to us was that o-CPG was complexing calcium ion. In order to determine if complex formation with calcium were taking place, conductometric titrations were performed. The titration curves of sodium hydroxide and of calcium hydroxide with o-CPG, glucuronolactone and salicylic acid, were compared. If the sodium hydroxide titration fits well to the calcium hydroxide titration curve, one concludes that no highly conductive ions are produced and complex formation is absent. The results showed that there is a possibility that a slight amount of complex formation may have occurred with glucuronolactone but certainly not with salicylic acid or o-CPG. Further proof that no complexing of calcium was taking place was obtained by measuring conductivity of separate solutions of calcium lactate and o-CPG at different concentrations. They were then combined, each at one half their original concentration and the resultant conductivity compared with the theoretical conductivity which we calculated. Results show that the combined conductivity is about the same as the theoretical and therefore complex formation is absent. This type of experiment was repeated using calcium chloride instead of calcium lactate and the same results were obtained. The only conclusion we can draw is that o-CPG does not complex calcium. It was thought that perhaps o-CPG complexes calcium and phosphate together. To test this hypothesis another dissolution experiment was designed similar to the first ones reported. However, the o-CPG was incubated in one case with phosphate and in another case with barium and phosphate before being added to the synthetic hydroxy apatite. The only test flask which showed a significant difference from the controls was that one in which o-CPG was incubated first with barium and phosphate. Here, the dissolution effect of o-CPG was decreased by 30 per cent. We conclude that calcium and phosphate may be complexed together by o-CPG. In summary we may state that o-CPG increases the dissolution of urinary tract calculi containing hydroxy apatite; it increases the dissolution of synthetic hydroxy apatite; it does not form a complex with calcium but it may form a complex with calcium and phosphate. The results also suggest that it may increase the dissolution of hydroxy apatite by acting at the surface of the crystal. This question requires further investigation.
Thesis (M.A.)--Boston University
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