Stesura Seveso Archivio Italiano di Urologia e Andrologia 2024; 96(4):13277 1 REVIEW The elimination half-life of theobromine is between 6 and 8 hours. The primary metabolites of theobromine are 3-methylxantine, 7-methylxantine, 7-methyluric acid and 3,7-dimethyluric acid, with 18-21% of theobromine excreted unchanged in the urine (3-5). Theobromine also derives from caffeine that is metabolized in the liver into paraxanthine (84%), theobromine (12%), and theophylline (4%). For this reason, individuals that non ingest theobromine (or chocolate) could have low, though detectable, urinary levels of theobromine (6, 7). Theobromine has stimulating action on the central nerv- ous system by intervening on the synapses, but this effect is weaker than caffeine (8). It has negligible toxicity in humans, because it is metab- olized very quickly. In other mammals, such as dogs and cats, it becomes toxic because they metabolize theo- bromine very slowly. The symptoms of intoxication are excitation, lowered heart rate, convulsions and in the most serious cases death (9). Theobromine has several various pharmacological appli- cations including cough suppression, increase of plasma HDL cholesterol and decrease of plasma LDL cholesterol, protection of enamel surface (10-13). A derivative of theobromine, 7-methylxanthine, has been used for the treatment of myopia (14). Theobromine should be administered as extract of cocoa beans rather than chocolate that contains large amounts of sugar and oxalate, that should be avoided in subjects with diabetes type 2 or metabolic syndrome and calcium oxalate renal stone formers. TREATMENT AND PREVENTION OF URIC ACID STONES Uric acid urinary stones account for about 10% of all the urinary stones although it is predictable an increase of their prevalence because of demographic and climate changes (15, 16). Uric acid stones form by crystallization of urinary uric acid when its concentration is above the threshold of sol- ubility which depends on pH. It ranges from 110 mg/L for urinary pH below 5.0 to 250 mg/L for pH 5.5 and up to 600 mg for a pH over 6.0. Urinary saturation for uric acid depends on urinary pH and urinary uric acid concentration (17-19). When urine is supersaturated with respect to uric acid, crystal forma- tion is possible, although above this threshold there is an interval of saturation values at which the solution is Theobromine (or 3,7-dimethylxanthine) is a natural alkaloid present in cocoa plant and its derivatives, such as chocolate. About 20% of ingested theo- bromine is excreted unchanged in the urine. Theobromine also derived from caffeine that is metabolized into theobromine by 12%. The primary metabolites of theobromine are 3-methylxan- tine, 7-methylxantine, 7-methyluric acid and 3,7-dimethyluric acid. Theobromine has an inhibitory activity of uric acid crystal- lization, because it has a structural pattern very similar to uric acid and can substitute uric acid molecules in the corresponding uric acid crystals, making them longer and thinner and decreas- ing their growth rate. Theobromine also favors the dissolution of crystals by decreasing supersaturation of uric acid by forming aggregates with uric acid through hydrogen bonds and aromatic stacking interactions (-stacking bonds) increasing urinary solu- bility of uric acid. Theobromine can be used for uric acid stone dissolution in combination with alkalinization to reduce the dose of citrate, thus preventing excessive alkalinization and the risk of formation of sodium urate crystals. Theobromine could also be used to treat patient with xanthine stones that cannot be dissolved by alkalinization because the solubility of xanthine is relatively independent of urinary pH. A metabolite of theo- bromine, 7-methylxanthine, has the potential to be used for the prevention of the formation of sodium urate crystals in the synovial fluid of gouty patients. KEY WORDS: Theobromine; Uric acid; Urinary calculi; Xanthine; Gout. Submitted 21 October 2024; Accepted 24 October 2024 INTRODUCTION Theobromine (or 3,7-dimethylxanthine) is a natural alka- loid present in Theobroma cacao (cocoa plant) and its derivatives. It is found in chocolate, and it is also present in small quantities in tea leaves (1, 2). Chemically it is a xanthine, a derivative of purine, whose related compounds include theophylline, caffeine, parax- anthine, and 7-methylxanthine, each of which differ in the number or placement of the methyl groups. Theobromine is derived from the nucleoside xanthosine by cleavage of the ribose and N-methylation to 7-methylxanthosine. Theobromine is slightly water-soluble but more fat-solu- ble, therefore requires 2-3 hours to peak, while caffein is highly water-soluble and peaks after only 30 minutes. In the liver, it is metabolized into xanthine and subse- quently into methyluric acid. Theobromine for treatment of uric acid stones and other diseases Alberto Trinchieri C.d.C. Ambrosiana, Cesano Boscone, Milan, Italy. DOI: 10.4081/aiua.2024.13277 Summary Archivio Italiano di Urologia e Andrologia 2024; 96(4):13277 A. Trinchieri 2 metastable that means that preformed crystals can grow but there is no formation of new crystals. Above the upper limit of the metastable zone of saturation crystals form spontaneously. Other factors can interfere with uric acid crystallization in the urine as the presence of hetero- geneous nuclei that can facilitate crystal formation or inhibitors of crystallization. Uric acid stones can be dis- solved when urinary saturation is under the threshold of supersaturation. Alkalinization Alkalinization by high doses of citrate and bicarbonate is commonly used for uric acid dissolution (20). Urinary pH should be raised over 6, although higher values are not rec- ommended because a urinary pH above 6.2 can cause the formation of an outer shell of insoluble calcium phosphate salts that are less soluble for higher pH values. Furthermore, in the presence of hyperuricosuria, deposits of sodium and/or potassium urate can form, because of reduced solu- bility of such urate salts at higher pH values (contrary to what happens for uric acid). Finally, long-term treatment with citrate can cause gastrointestinal disturbances. Other substances N-acetylcysteine (NAC) has been proposed for its alkalin- izing and mucolytic effect due to the cleavage of disulfide bridges of mucoproteins contained in the deposits of organic matter covering stone crystals (21, 22). In vitro studies suggested the use of some glycosamino- glycans, glycoproteins and saponins (such as ginseng extract) to interfere with crystallization of uric acid (23). However, these substances are not properly inhibitors because they act by modifying the surface tension of water and do not show dose-response relationships. THEOBROMINE More recently, in vitro studies showed that theobromine is a very effective inhibitor of uric acid crystallization (24, 25). This effect is clinically significant for urinary con- centration over 15 mg/L or higher, although higher con- centrations (80 mg/L) provided no additional benefit. Urinary concentrations in the therapeutical range are obtained after oral administration of 300 mg of theo- bromine with approximately 60 mg excreted unchanged in the urine. Caffeine, theophylline and paraxanthine showed no similar effects. In fact, they had a structure very similar to theobromine, but minimal modification of their chemical structure makes them ineffective as inhibitors. Theobromine inhibits nucleation of uric acid crystals being absorbed onto the faces of the crystals and modifying their morphology making them longer and thinner. In particular, theobromine may inhibit growth at only one of the faces of the crystal (210), but not at the others (001 and 201). Because uric acid and theobromine molecules have very similar structural patterns, theo- bromine can substitute uric acid molecules in the corre- sponding uric acid crystals. The incorporation of this molecule to the uric acid crystal lattice modifies the struc- ture of some layers so increasing their energy and decreasing their growth rate. In the presence of theo- bromine crystals formed on the surface of the uric acid were smaller. Thus, theobromine may be clinically useful in preventing the regrowth of uric acid calculi fragments. Theobromine also favors the dissolution of crystals by decreasing supersaturation of uric acid (26). The velocity of stone dissolution depends on the size and location of the stone and on flow of irrigation. Furthermore, the microstructure of the stone, as observed at scanning electronic microscopy can influence the process of dissolution. In fact, stones that appeared macroscopically similar, may have major differences in microstructure by the presence of porosities, the distribu- tion of organic matter and the size of crystals. The disso- lution can be made difficult by the presence of shells of sodium/potassium urate or apatite as consequence of very high pH (above 7) and high uric acid concentration (27). Combined treatment The combination of alkalization with citrate and theo- bromine patented and commercialized by the company Devicare in its Lit-Control pH Up treatment (28, 29), allows to use lower doses of citrate, thus preventing exces- sive alkalinization to avoid formation of sodium urate crystals (30). Furthermore, the reduced dose of potassium citrate administration decreases the risk of hyperkaliemia in patients with kidney failure or heart disease. THEOBROMINE AND XANTHINURIA Xanthinuria is a rare hereditary disorder related to a defi- ciency of xanthine dehydrogenase/oxidase (XDH/OX) caus- ing an accumulation of hypoxanthine and xanthine due to a reduced degradation of these two precursors to uric acid. This results in hypouricemia, hypouricosuria, xanthinuria and formation of xanthine urinary stones (31, 32). There are different types of xanthinuria due to mutations of different genes (33-35). The classical type I is caused by a mutation in XDH/XO gene mapped to chromosome 2p23.1. Type II depends on mutations in molybdenum cofactor sulfurase gene (MOCOS) localized on chromo- some 18q12.2 that cause a defect of XDH/OX and alde- hyde oxidase (AO). Triple deficiency of XDH, AOX and sulfite oxidase is caused by molybdenum cofactor defi- ciency type A (OMIM 252150) due to mutations in MOCS1 gene (6p21.1). The classical presentation occurs at any age with renal colic, hematuria and urinary tract infection associated to xanthine stones. Less frequently the presentation is more severe with renal failure, muscle-skeletal and gastroin- testinal symptoms. Traditional diagnosis with allopurinol loading test or liver biopsy has been replaced by genetic testing in stone patients with extremely low serum and low urinary uric acid replaced by xanthine. The treatment for patients with xanthinuria is a low purine diet and high intake of fluids. In contrast to patients with uric acid stones, urine alkalinization is not effective because the solubility of xanthine is relatively independent of urinary pH. A recent study showed that 1-methylxanthine (1-MX), 7-methylxanthine (7-MX), and 3-methylxanthine (3-MX) significantly inhibited xanthine crystallization in vitro in a concentration dependent manner (36). Archivio Italiano di Urologia e Andrologia 2024; 96(4):13277 3 Theobromine and uric acid stones Two of these molecules are major metabolites of theo- bromine whereas the third is a metabolite of caffeine. In fact, after theobromine ingestion, 20% is excreted as theo- bromine, 21.5% as 3-MX, and 36% as 7-MX and after consumption of caffeine, 19% of it is excreted as 1-MX. Hypoxanthine (HX), theophylline (TP), paraxanthine (PX), theobromine (TB), caffeine (CF), 1-methyluric acid (1-MU), and 1,3-dimethyluric acid (1,3-DMUA) showed no signifi- cant inhibitory effect on xanthine crystallization because only methyl derivatives of xanthine can be incorporated into the xanthine crystal lattice modifying its structure and slowing crystal growth (by increasing Gibbs free energy). However, even if theobromine did not inhibit xanthine crystallization by itself, it could be used for prevention of xanthine stones by the effects of its metabolites. THEOBROMINE AND GOUT Gout is a rheumatic disease presenting with pain, swelling, and redness in the peripheral joints, especially in the metatarsophalangeal joint in the big toe and other joints in the feet and hands (37). The disease is due to accumulation of monosodium urate (NaU) needle-shaped crystals in the affected joints. Synovial fluid is an ultrafiltrate from plasma with a pH of 7.4 and a sodium level of about 150 mmol/L. At a pH of 7.4 most uric acid is present as univalent anionic urate, whereas at serum urate level below 6 mg/dl sodium urate crystals do not form because sodium urate solubility threshold is 6.6 mg/dl. When urate levels are over the sol- ubility limit of 6.6 mg/dl sodium urate crystals start to form. Furthermore, solubility of sodium urate is related to temperature and tends to decrease at temperature lower than 37°C, being only 3.7 mg/dl at 26°C. This explains why crystal formation is more common in the joints of the hands and feet where temperature tends to be lower (38). The pathophysiology of crystal formation in synovial flu- ids is peculiar because the circulation of fluids in the cav- ities of synovial joints is quite limited with a much slow- er renewal of fluids compared to urinary tract where the flow of urine is continuous and relatively fast. Consequently, inhibitors that can prevent crystal forma- tion for a period up to 30-40 minutes can be useful in continuous flow of urine, but they are ineffective in absence of renewal of fluid as in the joint cavities. Treatment of gout is based on anti-inflammatory drugs and the reduction of serum urate levels by a low purine diet, or drugs decreasing the production of uric acid, as allopurinol and febuxostat or drugs increasing the urinary excretion of uric acid, such as probenecid. An alternative approach could be increasing urate solu- bility or inhibit sodium urate crystallization. Some in vitro studies demonstrated that the combination of arginine-rich peptide and copper ions was able to delay the crystallization of sodium urate (39, 40). Similarly, the addition of trimethoprim to urate solutions delayed sodi- um urate crystallization because trimethoprim acted as competing binding agent forming a more soluble co-crys- tal with sodium urate (41). Theobromine can form aggregates with uric acid through hydrogen bonds and aromatic stacking interactions (-stacking bonds) increasing urinary solubility of uric acid (42). Similarly, the solubility of uric acid increased in the presence of vitamin C (43). Furthermore, theo- bromine is also able to interact with uric acid crystals changing their morphology. Epidemiological studies demonstrated that coffee and chocolate consumption decreased the risk of gout (44, 45). An in vitro study suggested that 7-methylxanthine, a metabolite of theobromine, has the potential to be used for the prevention of gout (46). The same study found that 3-methylxanthine also prevented crystallization, although this happens at a four-fold-greater concentra- tion than with 7-methylxanthine; 7-methyluric acid has a slightly stronger effect than 3-methylxanthine, but its plasma levels are negligible. For this reason, consumption of 7-methylxanthine (or theobromine) has the potential for preventing the crystal- lization of sodium urate and the development of gout. Regarding the safety of the treatment of 7-methylxan- thine, studies documented that it has no toxic effects up to an oral dose of 1000 mg/kg of weight indicating that the consumption of 400 mg three times per day that should be requested to prevent gout seems to be safe. CONCLUSIONS Theobromine is a natural product that has several poten- tial therapeutic applications. The use for the treatment of uric stones is the most promising because it allows the dissolution of uric acid stones faster with the use of lower doses of alkalizers. However, clinical efficacy must be confirmed by randomized clinical trials. Treatment with theobromine or its derivatives could be used in the treat- ment of xanthinuria or gout. REFERENCES 1. Craig WJ, Nguyen TT. Caffeine and theobromine levels in cocoa and carob products. J Food Sci. 1984; 49:302-303. 2. Martínez-López S, Sarriá B, Gómez-Juaristi M, et al. Theobromine, caffeine, and theophylline metabolites in human plas- ma and urine after consumption of soluble cocoa products with dif- ferent methylxanthine contents. Food Res. Int. 2014; 63:446-455. 3. Gates S, Miners JO. Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism. Br J Clin Pharmacol. 1999; 47:299-305. 4. Tarka SM, Arnaud MJ, Dvorchik BH, Vesell ES. Theobromine kinet- ics and metabolic disposition. Clin Pharmacol Ther. 1983; 34:546-555. 5. Rodopoulos N, Höjvall L, Norman A. Elimination of theobromine metabolites in healthy adults. Scand J Clin Lab Invest. 1996; 56:373- 383. 6. Lelo A, Birkett DJ, Robson RA, Miners JO. Comparative pharma- cokinetics of caffeine and its primary demethylated metabolites paraxanthine, theobromine and theophylline in man. Br J Clin Pharmacol. 1986; 22:177-182. 7. Tang-Liu DS, Williams RL, Riegelman S. Disposition of caffeine and its metabolites in man. J Pharm Exp Ther. 1983; 224:180-185. 8. Mitchell ES, Slettenaar M, vd Meer N, et al. Differential contribu- tions of theobromine and caffeine on mood, psychomotor perform- ance and blood pressure. Physiol Behav. 2011; 104:816-822. 9. Finlay F, Guiton S. Chocolate poisoning. BMJ. 2005; 331:633. Archivio Italiano di Urologia e Andrologia 2024; 96(4):13277 A. Trinchieri 4 10. Halfdanarson TR, Jatoi A. Chocolate as a cough suppressant: rationale and justification for an upcoming clinical trial. Support Cancer Ther. 2007; 4:119-122. 11. Khan N, Monagas M, Andres-Lacueva C, et al. Regular con- sumption of cocoa powder with milk increases HDL cholesterol and reduces oxidized LDL levels in subjects at high-risk of cardiovascular disease. Nutr Metab Cardiovasc Dis. 2012; 22:1046-1053. 12. Neufingerl N, Zebregs YE, Schuring EA, Trautwein EA. Effect of cocoa and theobromine consumption on serum HDL-cholesterol con- centrations: a randomized controlled trial. Am J Clin Nutr. 2013; 97:1201-1209. 13. Kargul B, Ozcan M, Peker S, et al. Evaluation of human enamel surfaces treated with theobromine: a pilot study. Oral Health Prev Dent. 2012; 10:275-282. 14. Lai L, Trier K, Cui DM. Role of 7-methylxanthine in myopia prevention and control: A mini-review. Int J Ophthalmol. 2023; 16:969- 976. 15. Trinchieri A, Montanari E. Prevalence of renal uric acid stones in the adult. Urolithiasis. 2017; 45:553-562. 16. Brikowski TH, Lotan Y, Pearle MS. Climate-related increase in the prevalence of urolithiasis in the United States. Proc Natl Acad Sci U S A. 2008; 105:9841-9846. 17. Trinchieri A, Montanari E. Biochemical and dietary factors of uric acid stone formation. Urolithiasis. 2018; 46:167-172. 18. Wagner CA, Mohebbi N. Urinary pH and stone formation. J Nephrol. 2010; 23(Suppl 16):S165-169. 19. Grases F, Costa-Bauza A, Gomila I, et al. Urinary pH and renal lithiasis. Urol Res. 2012; 40:41-46. 20. Trinchieri A, Esposito N, Castelnuovo C. Dissolution of radiolu- cent renal stones by oral alkalinization with potassium citrate/potas- sium bicarbonate. Arch Ital Urol Androl. 2009; 81:188-91. 21. Burns JR, Gauthier JF, Finlayson B. Dissolution kinetics of uric acid calculi. J Urol. 1984; 131:708-711. 22. Sadowska AM. N-acetylcysteine mucolysis in the management of chronic obstructive pulmonary disease. Ther Adv Respir Dis. 2012; 6:127-135. 23. Grases F, Ramis M, Villacampa AI, Costa-Bauzá A. Uric acid urolithiasis and crystallization inhibitors. Urol Int. 1999; 62:201- 204. 24. Grases F, Rodriguez A, Costa-Bauza A. Theobromine inhibits uric acid crystallization. A potential application in the treatment of uric acid nephrolithiasis. PLoS ONE. 2014; 9:e111184. 25. Costa-Bauza A, Grases F, Calvó P, et al. Effect of Consumption of Cocoa-Derived Products on Uric Acid Crystallization in Urine of Healthy Volunteers. Nutrients. 2018; 10:1516. 26. Chattaraj KG, Paul S. Inclusion of theobromine modifies uric acid aggregation with possible changes in melamine-uric acid clusters responsible for kidney stones. J Phys Chem B. 2019; 123:10483-10504. 27. Sadi MV, Saltzman N, Feria G, Gittes RF. Experimental obser- vations on dissolution of uric acid calculi. J Urol. 1985; 134:575-579. 28. Grases F, et al. Combination of a urinary basifying agent and an uric acid crystallisation inhibitor for the treatment and prevention of renal lithiasis. EP3130337B1, 2019. 29. Grases F, et al. Theobromine or its derivatives for the treatment or prevention of uric acid renal lithiasis. EP3150208B1, 2023. 30. Hernandez Y, Costa-Bauza A, Calvó P, et al. Comparison of two dietary supplements for treatment of uric acid renal lithiasis: citrate vs citrate + theobromine. Nutr. 2020; 12:2012. 31. Carpenter TO, Lebowitz RL, Nelson D, Bauer S. Hereditary xan- thinuria presenting in infancy nephrolithiasis. J Pediatr. 1986; 109:307-309. 32. Mateos FA, Puig JG, Jimenez ML, Fox IH. Hereditary xanthin- uria: Evidence for enhanced hypoxanthine salvage. J Clin Invest. 1987; 79:847-852. 33. Ichida K, Amaya Y, Okamoto K, Nishino T. Mutations associat- ed with functional disorder of xanthine oxidoreductase and heredi- tary xanthinuria in humans. Int J Mol Sci. 2012; 13:15475-15495. 34. Ichida K, Matsumura T, Sakuma R, et al. Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xan- thinuria type II. Biochem Biophys Res Commun. 2001; 282:1194-1200. 35. Mraz M, Hurba O, Bartl J, et al. Modern diagnostic approach to hereditary xanthinuria. Urolithiasis. 2015; 43:61-67. 36. Grases F, Costa-Bauza A, Roig J, Rodriguez A. Xanthine urolithi- asis: Inhibitors of xanthine crystallization. PLoS ONE. 2018; 13:e0198881. 37. Archer HE, Rhoden E. Uric-acid levels in blood and plasma. Br Med J. 1951; 2:947. 38. Kippen I, Klinenberg JR, Weinberger A, Wilcox WR. Factors affecting urate solubility in vitro. Ann Rheum Dis. 1974; 33:313-317. 39. Liu Y, Zhang Q, Du J, Guo R. Arginine-rich peptides as crystal- lization inhibitors for sodium urate. J Mater Chem B. 2023; 11:7389- 7400. 40. Liu Y, Zhang Q, Li N, et al. Efficient synergistic cooperation of an arginine-rich peptide and copper ions in sodium urate crystallization inhibition. Langmuir. 2023; 39:9942-9951. 41. Hall VM, Thornton A, Miehls EK, et al. Uric acid crystallization interrupted with competing binding agents. Cryst. Growth Des. 2019; 19:7363-7371. 42. Chattaraj KG, Paul S. Appraising the potency of small molecule inhibitors and their graphene surface-mediated organizational attributes on uric acid-melamine clusters. Phys Chem Phys. 2022; 24:1029-104. 43. Chattaraj KG, Paul S. The miscibility and solubility of uric acid and vitamin C in the solution phase and their structural alignment in the solid-liquid interface. Phys Chem Chem Phys. 2021; 23:15169- 15182. 44. Choi HK, Curhan G. Coffee consumption and risk of incident gout in women: The Nurses’ Health Study. Am J Clin Nutr. 2010; 92:922-927. 45. Dillinger TL, Barriga P, Escárcega S, et al. Food of the gods: Cure for humanity? A cultural history of the medicinal and ritual use of chocolate. J Nutr. 2000; 130(Suppl. S8):2057S-2072S. 46. Costa-Bauza A, Grases F. 7-methylxanthine inhibits the forma- tion of monosodium urate crystals by increasing its solubility. Biomolecules. 2023; 13:1769. Correspondence Alberto Trinchieri, MD (Corresponding Author) alberto.trinchieri@gmail.com C.d.C. Ambrosiana, Cesano Boscone, Milan, Italy Conflict of interest: The authors declare no potential conflict of interest.