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Article

Effect of Methylxanthines on Urate Crystallization: In Vitro Models of Gout and Renal Calculi

by
Jaume Dietrich
,
Felix Grases
and
Antonia Costa-Bauza
*
Laboratory of Renal Lithiasis Research, University Institute of Health Sciences Research (IUNICS), University of Balearic Islands, 07122 Palma de Mallorca, Spain
*
Author to whom correspondence should be addressed.
Crystals 2024, 14(9), 768; https://doi.org/10.3390/cryst14090768
Submission received: 19 July 2024 / Revised: 22 August 2024 / Accepted: 28 August 2024 / Published: 29 August 2024
(This article belongs to the Special Issue Pathological Biomineralization: Recent Advances and Perspectives)
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Abstract

:
Background: Common forms of pathological crystals are uric acid or urates, which are responsible for gout, urolithiasis, and other conditions. Methods: We used a kinetic–turbidimetric crystallization assay to evaluate the effect of ten specific methylxanthines on the crystallization of monosodium urate, potassium urate, and ammonium urate in conditions that mimicked urine. We also studied the effect of different levels of 7-methylxanthine in the presence of other biological compounds (albumin and hyaluronic acid) on the solubility of monosodium urate in conditions that mimicked synovial fluid. Results: The results showed that 7-methylxanthine in the range of 16.61–49.84 mg/L inhibited the crystallization of each urate when the initial urate concentration was 3 × 10−3 M (500 mg/L) and the conditions mimicked urine, and that the greatest inhibitory effect was for monosodium urate. In addition, 7-methylxanthine at a concentration of 25 mg/L totally prevented the crystallization of monosodium urate at an initial urate concentration of 2.38 × 10−3 M (400 mg/L) in conditions that mimicked synovial fluid. Moreover, at a low concentration of 7-methylxanthine, albumin and hyaluronic acid increased this inhibitory effect. Conclusions: Our in vitro results demonstrate that 7-methylxanthine inhibits the crystallization of urates in conditions that mimic synovial fluid and urine.

1. Introduction

Common forms of pathological crystals are uric acid or urates, which are responsible for gout, urolithiasis, and other conditions. Uric acid mainly crystallizes as a pure anhydrous or a dihydrate form, and different morphologies of the crystalline uric acid can occur in urinary stones, such as the pseudorhombic and monoclinic forms [1]. Uric acid usually forms round stones with an interior consisting of concentric layers of crystalline material that forms radial laminations, and less commonly with an interior that is granular [2]. Uric acid is a constituent of urinary stones that often have other components, such as calcium oxalate and different urates. Ammonium urate is the most common urate to precipitate in urinary stones, and it is more commonly found in the presence of struvite. Potassium and sodium urates can also occur in urinary stones, but calcium urate is a rare component of urinary stones in humans. Some studies reported the presence of non-stoichiometric urate stones that contain various cations in certain areas that are near the uric acid, ammonium urate, and sodium urate crystals [3].
The precipitation of different types of uric acid depends on the conditions. Anhydrous uric acid is more thermodynamically stable, and dihydrate uric acid tends to gradually transition into the anhydrous form in vitro and in vivo. Thus, anhydrous uric acid is the most prevalent phase in uric acid stones.
Numerous factors can influence the crystallization of urate salts in physiological conditions. Crystallization occurs through the nucleation of molecules that surpass the dispersion forces imposed by the local environment and begins with the formation of clusters. Temperature also has an important role, in that the solubility of monosodium urate decreases from 6.8 to 6.0 mg/dL when the temperature decreases from 37 °C to 35 °C [4]. This is part of the reason why monosodium urate crystallization in gout occurs in distal articulations, such as the big toe, where body temperatures are slightly lower. The pH also plays an important role in urate salts’ crystallization, even though it does not greatly impact their solubility [5]. Endogenous factors can also affect crystallization, in that proteins, collagen fibers, and other organic macromolecules often promote urate crystallization [6]. Many studies have examined the effect of albumin on the crystallization of urate salts, but the results have been discordant. Some studies reported that albumin inhibited crystallization [7], and other studies reported that it acted as a nucleating agent by enhancing cluster formations due to the interaction of its external carboxylate groups with sodium ions [8]. There is also evidence that certain physiological conditions alter the effect of albumin on crystallization.
Hyperuricuria and certain other conditions are the principal factors that promote urate salts’ crystallization in urine. An increase in urinary pH above 7 leads to urate univalent anion being the predominant form of uric acid. Thus, a high urinary pH and an elevated concentration of urinary urate can lead to a urate salts’ supersaturation and the formation of the corresponding renal stones.
The presence of endogenous and exogenous crystallization inhibitors in vivo can prevent pathological crystallizations. Some of these inhibitors, which can be organic or inorganic compounds, can retard or totally prevent the formation of crystals. Thus, many studies have examined the effects of different inhibitors on the formation of urate crystals. For example, two dyes, Bismarck brown (at 25 mg/L) and methylene blue (at 100 mg/L), were found to be crystal growth inhibitors of monosodium urate [9]; concentrations above 1.0 mg/L of 1-methyluric acid displayed a 60% inhibition of ammonium urate crystal growth and poly(ethyleneimine) completely suppressed it [10]; and protein polysaccharides of connective tissue (PPL) at a concentration of 8 mg/mL increased the solubility of monosodium urate from 5.7 mg/100 mL to 25.5 mg/100 mL [11]. Vegetable extracts can also affect monosodium urate crystallization in vitro, and a study that examined an aqueous extract of 1% of Rotula aquatica and Aerva lanata reported that this extract significantly inhibited crystal formation [12]. Nevertheless, most of these substances are unsuitable for the treatment of pathologies related to urate crystallization because they typically have insufficient inhibitory capacity or do not reach active concentrations in biological fluids after oral administration. In the case of gout, xanthine oxidase inhibitors, which inhibit the production of uric acid or the increase in uric acid urinary excretion using uricosuric agents, are often used to prevent the formation of monosodium urate crystals [13]. Although methylene blue and Bismarck brown have also been described as inhibitors of the crystallization of monosodium urate crystals [9], any paper in the bibliography reporting their clinical application cannot be found.
Epidemiological studies showed that coffee intake was associated with a decreased prevalence of gout [14]. This effect is attributed to caffeine, whose purine structure is similar to that of urate, thus suggesting that caffeine may effectively interact with urate. Our recent study evaluated the effects of caffeine, theobromine, and some of their most common metabolites on the crystallization of monosodium urate [15]. Surprisingly, the results showed that in the studied conditions, neither caffeine nor theobromine inhibited the crystallization of monosodium urate in conditions that resembled synovial fluid, but that 7-methylxanthine (a metabolite of both compounds) totally prevented crystallization.
In this paper, we further evaluated the effect of 7-methylxanthine on the crystallization of monosodium urate, studying the influence of different substances present in synovial fluid, such as calcium, albumin, and hyaluronic acid, as well as the effects of 7-methylxanthine on the crystallization of monosodium urate in real human plasma. Moreover, the effects of different methylxanthines on the crystallization of the three most common urate salts found in renal calculi in conditions that mimic urine have also been studied.

2. Materials and Methods

2.1. Reagents and Solutions

Uric acid, caffeine, theophylline, theobromine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, 1,3-dimethyluric acid, pentoxifylline, etophylline, and dyphylline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Synthetic urine components were obtained from Panreac (Montcada i Reixac, Barcelona, Spain). Chemicals of analytical- or reagent-grade purity were dissolved in ultra-pure deionized water from a Milli-Q system (Merck-Millipore, Darmstadt, Germany). Then, 2 mM solutions of dyphylline, caffeine, theophylline, theobromine, pentoxifylline, etophylline, 1,3-dimethyluric acid, 1-methylxanthine, and 3-methylxanthine (Figure 1) were prepared; a 1.9 mM solution of 7-methylxanthine was prepared due to its low solubility in water. The addition of a few drops of 0.1 M NaOH (as necessary) was used to dissolve the different methylxanthines. The low solubility of 7-methylxanthine necessitated several hours of stirring with heat to achieve dissolution. After preparation, all solutions were stored at room temperature.
A uric acid stock solution (2 g/L) was prepared daily by dissolving 1 g of uric acid in 0.5 L of water, followed by the addition of 3 M NaOH to adjust the pH to 10.70.
For in vitro crystallization studies of ammonium urate, monosodium urate, and potassium urate, synthetic urine stock solutions were prepared with an elevated concentration of each cation to promote crystallization (Table 1). The synthetic urine was prepared without calcium or oxalate (to prevent calcium oxalate crystallization) and without magnesium (to prevent ammonium magnesium phosphate crystallization).
Sodium urate crystallization was performed in conditions that mimicked the synovial fluid: phosphate-buffered saline (PBS) containing 12 mM phosphate buffer, 400 mM Na+, and a uric acid concentration of 300 to 475 mg/L (1.79 to 2.84 mM) at pH 7.41.

2.2. Effect of Different Methylxanthines on Crystallization of Urate Salts in Artificial Urine

A previously described turbidimetric assay was adapted to determine the effect of methylxanthines on the kinetics of the crystallization of urate salts in the synthetic urine medium at pH 7.4 to 7.5 [16]. This medium contained 500 mg/L of uric acid for experiments with ammonium urate and monosodium urate and contained 750 mg/L of uric acid for experiments with potassium urate. These conditions were designed to achieve optimal induction times for the crystallization of the different urate salts. In other words, the crystallization time was not too short, because high supersaturation would prevent the inhibitory effect of methylxanthines; the crystallization time was not too long because this would decrease the reproducibility of the assays. This timing was also adjusted to match the typical residence time of urine in the urinary tract.
The turbidimetric system consisted of a photometer (AvaSpec-ULS2048CL-EVO, Avantes, The Netherlands) that was equipped with a fiber-optic light-guide and a measuring cell that was attached to a light path (2 × 10 mm) by a reflector. This instrument was operated in the kinetic mode, and absorbance measurements were integrated from 400 to 600 nm. Crystallization was assessed at a constant temperature (37 °C) with mixing by a magnetic stir bar (250 rpm). More specifically, 20 mL of water and 10 mL of a 2 g/L uric acid solution (for ammonium urate and monosodium urate experiments) or 15 mL of water and 15 mL of a 2 g/L uric acid solution (for potassium urate experiments) were added to a crystallization flask, followed by the addition of 10 mL of the synthetic urine stock solution. Then, 0.2 to 0.3 mL of 1 M NaOH was added until the pH reached 7.4 to 7.5, conditions that cause the supersaturation of urate. Absorbance was recorded continuously to monitor crystal formation, and the time when absorbance first increased was considered to be the time of crystal induction (ti). Each experiment was performed in triplicate.
In the experiments with the methylxanthines, a volume necessary to obtain a final concentration of 0.3 mM (with a corresponding reduction in the volume of added water to reach a final volume of 40 mL in every replicate) was added from the prepared stock solution of each methylxanthine, and the solution was stirred for 15 min to promote the interaction of the urate and methylxanthine before the addition of artificial urine and adjustment of the pH. Lower concentrations and possible synergistic effects were also assessed for methylxanthines that had inhibitory effects by adding different concentrations of two compounds into the same solution. Crystallization times were measured as described above.
The maximum tested concentration of methylxanthines (0.3 mM) was selected based on previous studies in which the urinary concentrations of different methylxanthines were determined after the consumption of cocoa products or theobromine (120 mg/day, for 14 days). In that study, the highest detected urinary concentration of 7-methylxanthine was around 0.3 mM [17].
Crystals that formed during this assay were also collected by passing the solution through a filter that had 0.45 µm pores. These crystals were then dried in a desiccator and examined by scanning electron microscopy (SEM).

2.3. Effect of 7-Methylxanthine on Crystallization of Monosodium Urate in Conditions That Mimic Synovial Fluid

The effect of 7-methylxanthine on the crystallization of monosodium urate in conditions that mimicked synovial fluid was examined in polystyrene non-tissue culture 12-well plates (Corning, Durham, NC, USA), each of which contained 5 mL of a crystallizing solution. These plates were sealed with acetate tape (Corning, Durham, NC, USA) to prevent evaporation, as previously reported [15]. The effect of different levels of sodium (150–400 mM), uric acid (70–370 mg/L), calcium (0 or 96 mg/L), albumin (0 or 10 g/L), and hyaluronic acid (0 or 3 g/L) on the ability of 7-methylxanthine to prevent sodium urate crystallization was assessed.
These crystallization experiments were performed in a PBS solution containing 12 mM phosphate buffer at pH 7.4. The plates were stored for 96 h at 31 °C, and the effects of different conditions on sodium urate crystallization were evaluated by measuring the amount of urate remaining in solution based on absorbance at 294 nm (see below) after appropriate dilution. All experiments were performed in triplicate, and the results are reported as means with standard errors.
The effect of different concentrations of 7-methylxanthine (0, 20, 40, 60, 80, 100, 125, 150, 200, 250, and 300 µM) on the crystallization of sodium urate in the presence of 400 mg/L of initial uric acid was determined. For these experiments, a solution containing 42 mL of AU 1 g/L, 10.5 mL of 0.12 M monosodium phosphate, 27.153 mL of 1.5 M sodium chloride, and 8.778 mL of water was prepared. After adjustment to pH 7.4, 5.474 mL of this solution was added into each well of a plate that contained 1.026 mL of water (control) or an appropriate volume of 1.9 mM of 7-methylxanthine with water to achieve the desired concentration and a final volume of 6.5 mL.
The effect of 50 µM of 7-methylxanthine on the crystallization of different initial concentrations of uric acid was also evaluated. In these experiments, 70 mg/L of uric acid and 50 µM of 7-methylxanthine were first added to the solution. Then, different amounts of uric acid were added to achieve final concentrations of 0, 100, 150, 200, 225, 250, 275, and 300 mg/L. The controls received the same treatment but without 7-methylxanthine.
The effect of the initial concentration of sodium on sodium urate crystallization in the presence of different concentrations of 7-methylxanthine was also studied. The sodium concentration in these experiments was 400, 250, 170, or 150 mM; the initial concentration of uric acid was 400 mg/L; and all other conditions were the same as described above.
The effect of 2.4 × 10−3 M (96 mg/L) Ca2+, 10 g/L of albumin (Sigma-Aldrich, St. Louis, MO, USA), and 3 g/L of two different hyaluronic acids (one from Bioibèrica, Barcelona, Spain; one from Sigma-Aldrich) in the presence of 7-methylxanthine in the concentration range of 0–300 µM on the crystallization of sodium urate in the presence of 400 mg/L of initial uric acid with 400 mM of sodium was also studied.

2.4. Effect of 7-Methylxanthine on Crystallization of Monosodium Urate in Human Plasma

Additional experiments used human plasma from healthy volunteers (purchased from Medpace, Madrid, Spain) to determine the effect of 7-methylxanthine on sodium urate crystallization. Prior to these experiments, plasma samples were collected from the supernatant after centrifugation for 10 min at 4200 rpm of supplied samples, containing a normal level of plasmatic uric acid.
These experiments were carried out in 96-well plates (Brand GMBH, Wertheim, Germany) with smaller working volumes, because of the limited availability of human plasma. Initial studies using PBS confirmed that the results obtained with 96-well plates were not significantly different from those obtained with 12-well plates.
First, 12 wells (3 columns of 4 wells each) were prepared in a 96-well plate. Then, 290 µL of the plasma supernatant was added into each well. In the first well of each column, 40 µL of Milli-Q water was added. In the second well of each column, 40 µL of uric acid (0.438 g/L) was added to achieve an increase in uric acid concentration of 50 mg/L. In the third well of each column, 40 µL of uric acid (0.875 g/L) was added to increase the uric acid concentration in 100 mg/L. In the fourth well of each column, 40 µL of uric acid (1.75 g/L) was added to achieve an increase in uric acid concentration of 200 mg/L. Then, in the 4 wells of the first column (controls without 7-metylxanthine), 20 µL of Milli-Q water was added; 20 µL of 7-methylxanthine (0.875 mM) was added to the 4 wells of the second column to achieve a final concentration of 50 µM; 20 µL of 7-methylxanthine (1.75 mM) was added to the 4 wells of the third column to achieve a final concentration of 100 µM. Then, the plate was sealed and incubated at 31 °C for 96 h. After that time, the plate was taken out and the amount of urate remaining in solution was determined by the uricase method (see below) after appropriate dilution.

2.5. Determination of Uric Acid

For experiments performed in PBS solutions, the concentration of urate remaining in solution at the end of the 96 h incubation period was quantified using a spectrophotometric assay [18], in which absorbance at 294 nm was proportional to the concentration of uric acid. A standard curve was established using uric acid standards (10 to 60 mg/L) in 1.92 mM phosphate buffer and 64 mM Na+. Prior measurements confirmed that 7-methylxanthine did not interfere in this assay.
For experiments that examined plasma, the concentration of urate remaining in the sample after 96 h was quantified using a uricase kit (Spinreact, Girona, Spain). The simpler spectrophotometric test could not be used due to the presence of organic compounds in plasma that also absorb UV radiation.

2.6. Scanning Electron Microscopy of Crystals

Crystals obtained from the kinetic–turbidimetric assays were collected by vacuum filtration after passage through a filter with 0.45 µm pores. These crystals were placed on a sample holder, fixed with an adhesive conductive tape, and then observed with a scanning electron microscope (SEM; TM4000 Plus II, Hitachi, Tokyo, Japan) for the assessment of morphology.

3. Results

3.1. Crystallization of Different Urates in Artificial Urine

We used an established kinetic–turbidimetric assay to measure crystallization under different conditions in artificial urine. Thus, we first selected adequate crystallization times for the controls (no methylxanthines) based on the typical residence time of urine in the urinary tract. Without methylxanthines, ammonium urate crystallized in 4.6 ± 0.4 min, monosodium urate crystallized in 5.7 ± 0.5 min, and potassium urate crystallized in 8 ± 0.7 min.
We then evaluated the effect of 10 different methylxanthines (0.3 mM) on the crystallization time of ammonium urate, monosodium urate, and potassium urate relative to the controls without methylxanthines (Figure 2). The effects of each methylxanthine are reported as the increment in induction time (∆ti) with respect to the ti without methylxanthines.
The results show that at 0.3 mM, 3-methylxanthine and 7-methylxanthine led to significantly longer crystallization times of ammonium urate (∆ti 3-MX = 9.33 ± 2.1 min, ∆ti 7-MX = 6.65 ± 2.3 min), monosodium urate (∆ti 3-MX = 4.4 ± 1.43 min, ∆ti 7-MX = 40 ± 6.4 min), and potassium urate (∆ti 3-MX = 6.4 ± 1.4 min, ∆ti 7-MX = 7.25 ± 0.48 min). The other methylxanthines were less effective inhibitors.
The supersaturation ratio for each urate salt (ammonium, sodium, or potassium) in the corresponding crystallizing solution was calculated using the following formula:
SS = [A−]x ϒA [B+]y ϒB/Ksp
where A corresponds to urate, B corresponds to the cation, Ksp is the thermodynamic solubility product of the corresponding urate salt, and ϒA and ϒB are the activity coefficients of the ions at the ionic strength (I) of the solution, calculated using the Debye–Hückel equation.
As can be seen in Table 2, of the three urate salts studied, ammonium urate is the least soluble, and interestingly, for this substance, 3-methylxanthine is more of an inhibitor than 7-methylxanthine, while 7-methylxanthine is more of an inhibitor for monosodium urate in this medium.
We then examined the effect of different concentrations of 3-methylxanthine and 7-methylxanthine on the crystallization of the different urates (Figure 3). The results show that an increased concentration of these two methylxanthines increased the crystallization time of each urate and that this effect was greatest for 7-methylxanthine with monosodium urate.
We then examined the combined effect of 3-methylxanthine and 7-methylxanthine on the crystallization time of ammonium urate (Figure 4A) and potassium urate (Figure 4B).
The results show that for a given concentration of one inhibitor, the crystallization time increased as the concentration of the other inhibitor increased. Moreover, the effect of these two inhibitors was additive, in that the crystallization time of a mixture was about the same as the sum of the crystallization times of each individual inhibitor at the same concentration. A synergistic effect of these two inhibitors would manifest as a greater slope of the curve for the increase in crystallization time vs. concentration with respect to the presence of only one inhibitor.

3.2. Effect of 7-Methylxanthine on Crystallization of Monosodium Urate in Conditions That Mimic Synovial Fluid

We then measured the effect of 7-methylxanthine on the crystallization of monosodium urate in conditions that mimicked synovial fluid (Figure 5). The results show that crystallization decreased as the concentration of 7-methylxanthine increased and that 150 µM (25 mg/L) of 7-methylxanthine totally inhibited the crystallization of monosodium urate under these conditions.
We then compared the effect of different initial concentrations of uric acid in samples without 7-methylxanthine and with 50 µM of 7-methylxanthine (Figure 6). The results show that 50 µM of 7-methylxanthine totally inhibited monosodium urate crystallization when the initial uric acid concentration was 225 mg/L or lower. In contrast, crystallization occurred at lower initial uric acid concentrations (threshold ~175 mg/L) in the absence of 7-methylxanthine.
We also examined the effect of 7-methylxanthine concentration (0–300 µM) and sodium concentration (150–400 mM) in the presence of 400 mg/L of the initial concentration of uric acid on the crystallization of monosodium urate (Figure 7). The results show that there was less crystallization as the sodium concentration decreased. Thus, for a sodium concentration of 170 mM, 25 µM of 7-methylxanthine totally inhibited monosodium urate crystallization. However, for a sodium concentration of 400 mM, 150 µM of 7-methylxanthine was needed to totally inhibit monosodium urate crystallization.
We performed a similar experiment to determine the effect of calcium on monosodium urate crystallization (Figure 8). The results show that calcium ions seemed to slightly reduce the inhibitory effect of 7-methylxanthine on monosodium urate crystallization. However, this effect was small, and a slightly greater level of 7-methylxanthine was sufficient to overcome the effect of calcium.
We performed similar experiments to determine the effect of albumin and hyaluronic acid (common components of synovial fluid) on monosodium urate crystallization (Figure 9). Albumin decreased monosodium urate crystallization when the concentration of 7-methylxanthine was below 100 µM and also inhibited monosodium urate crystallization in the absence of 7-methylxanthine. However, albumin slightly promoted monosodium urate crystallization when the concentration of 7-methylxantine was greater than 150 µM.
Our examination of two commercial hyaluronic acid products showed that each one inhibited monosodium urate crystallization alone and in the presence of 7-methylxanthine and that the product from Bioibèrica had a greater inhibitory effect (Figure 9). More specifically, the Bioibèrica product almost totally inhibited monosodium urate crystallization when the concentration of 7-methylxanthine was 40 µM or more. Similar to albumin, the Sigma-Aldrich product slightly promoted crystallization when the concentration of 7-methylanthine was greater than 150 µM.
We then performed crystallization experiments with human plasma (Figure 10). This plasma had an initial uric acid concentration of 34.38 mg/L (determined in the basal well without the addition of uric acid). As expected, in the absence of 7-methylxanthine, monosodium urate crystallization increased as the concentration of uric acid increased up to 250 mg/L. The addition of 50 µM of 7-methylxanthine totally inhibited monosodium urate crystallization at uric acid concentrations lower than 130 mg/L, and the addition of 100 µM of 7-methylxanthine totally inhibited crystallization at all tested concentrations of uric acid.

3.3. Crystal Morphology under Different Conditions

We used SEM to observe the morphology of urate crystals that formed without methylxanthines, with 0.3 mM 3-methylxanthine, and with 0.3 mM 7-methylxanthine in urinary conditions (Figure 11, Figure 12 and Figure 13).
Taken together, these figures show slight differences in crystal morphology in the absence and presence of these two methylxanthines. Notably, the addition of each methylxanthine led to the formation of thinner and smaller crystals.

4. Discussion

Consistent with previous studies, we found that 7-methylxanthine inhibited the crystallization of monosodium urate in conditions that mimicked synovial fluid. We also extended these findings by examining the crystallization of three different urates in conditions that mimicked urine. Some tested methylxanthine compounds in our experiments with synthetic urine are metabolites of dietary methylxanthines that occur in the body [20]. However, we excluded some other metabolites due to their low concentrations in plasma and urine [21].
Our initial kinetic–turbidimetric assays in synthetic urine allowed us to identify the specific methylxanthines that inhibited the crystallization of ammonium urate, monosodium urate, and potassium urate. Among the studied methylxanthines, 3-methylxanthine and 7-methylxanthine were the most potent inhibitors of crystallization. After the ingestion of cacao products, the concentration of 3-methyxanthine was 0.6 µM in plasma and 34.8 µM in urine; the corresponding numbers for 7-methylxanthine were 2.1 µM (plasma) and 110.1 µM (urine) [22]. These concentrations increased the time for the induction of crystallization in all three of the urate salts we examined. However, the effect of these two methylxanthines depended on the specific urate salt, pointing to the relevance of the corresponding cation. For ammonium urate and potassium urate, the two methylxanthines had similar effects at the studied conditions, but for monosodium urate, 7-methylxanthine had a 10-fold greater inhibition of crystallization.
The effect of 7-methylxanthine on the crystallization of monosodium urate in conditions that mimicked urine is concordant with its observed effects in conditions that mimicked synovial fluid [15]. This finding points toward the possible future use of 7-methylxanthine for preventing the formation of monosodium urate kidney stones.
Our SEM observations showed that the different methylxanthines led to smaller crystals and slight variations in crystal morphology, although these morphological differences were relatively minor. The slight methylxanthine-induced morphological changes in these crystals are presumably related to their effect on crystallization time. Nonetheless, the effect of these inhibitors was clearly thermodynamic, because clusters consisting of the inhibitor and the urate ion increased the solubility of the urate and therefore decreased the supersaturation and prevented precipitation of the complexed urate ions [15,23].
In our experiments that mimicked synovial fluid (uric acid concentration: 2.38 × 10−3 M (400 mg/L), NaCl concentration: 4 × 10−1 M), we observed that 1.5 × 10−4 M (25 mg/L) 7-methylxanthine totally prevented the crystallization of monosodium urate. When the sodium concentration decreased, supersaturation also decreased, and this also decreased the crystallization of monosodium urate. In fact, at NaCl concentrations lower than 1.7 × 10−1 M, no monosodium urate crystallization was observed. Thus, the lower the sodium concentration, the lower the concentration of 7-methylxanthine needed to completely prevent monosodium urate crystallization.
The effect of albumin on the crystallization of urates has been controversial since the first studies examined this effect [8], and it is still uncertain whether albumin inhibits or promotes crystallization. Some studies concluded that albumin functions as an inhibitor or a nucleant depending on its concentration and pH [8]. However, we observed that albumin and hyaluronic acid inhibited the crystallization of monosodium urate at low concentrations of 7-methylxanthine. Moreover, in the absence of 7-methylxanthine, albumin and hyaluronic acid also had slight inhibitory effects, suggesting that they may directly interact with some components of monosodium urate, and not with 7-methylxanthine. We also found that 7-methylxanthine was an effective inhibitor of monosodium urate crystallization in blood plasma. Under these conditions, with a uric acid concentration of 1.19 × 10−3 M (200 mg/L), 1.0 × 10−4 M (16.6 mg/L) 7-methylxanthine totally prevented monosodium urate crystallization.
The effects of 7-methylxanthine on the nucleation of monosodium urate, analogous to the effects of theobromine on the crystallization of uric acid [16], can be explained as a consequence of two phenomena: on the one hand, a kinetic effect when adsorbed on the surface of the crystals in formation, hindering their development, and on the other hand, as a consequence of a thermodynamic effect, due to the formation of stable clusters (formation of adducts by hydrogen bonds) in solution between urate ions and 7-methylxanthine analogous to theobromine–UA complexes [23], which generates an increase in the solubility of monosodium urate with the subsequent decrease in the supersaturation of monosodium urate in the solution; so, by decreasing the driving force of crystallization, its crystallization is prevented or hindered.
It is interesting to consider that theobromine is an efficient crystallization inhibitor and solubility enhancer of uric acid in urine [16]. When it is orally ingested, 21% is excreted as theobromine, but 36% is excreted as 7-methylxanthine [24], which can act as a solubility enhancer of monosodium urate. 7-methylxanthine is one of the products of metabolism of caffeine and theobromine; specifically, 8% of an oral dose of caffeine (consumed as coffee) is metabolized to 7-methylxanthine [25], and 30% of an oral dose of theobromine (consumed as dark chocolate) is metabolized to 7-methylxanthine [25]. In addition, 7-methylxanthine has several pharmacological applications, including relief of asthma symptoms, as it has bronchodilator properties, and recently, the use of 7-methylxanthine for the treatment of myopia has been studied [26,27].

5. Conclusions

Among the 10 methylxanthines we studied, 3-methylxanthine and 7-methylxanthine were the strongest inhibitors of the crystallization of the three examined urate salts in artificial urine, and the strength of this inhibition depended on the cation. The strongest inhibitory effect was for 7-methylxanthine on the crystallization of monosodium urate (∆ti = 40 min). In conditions that mimicked synovial fluid, 1.5 × 10−4 M (25 mg/L) of 7-methylxanthine totally inhibited the crystallization of monosodium urate at an initial uric acid concentration of 2.38 × 10−3 M (400 mg/L). Moreover, certain biomolecules (albumin and one of the two tested hyaluronic acids) increased inhibition at low levels of 7-methylxanthine but decreased inhibition when the level of 7-methylxanthine was above 150 µM.

Author Contributions

Conceptualization, F.G. and A.C.-B.; methodology, J.D., F.G. and A.C.-B.; investigation, J.D.; resources, F.G. and A.C.-B.; writing—original draft preparation, J.D., F.G. and A.C.-B.; writing—review and editing, J.D., F.G. and A.C.-B.; funding acquisition, F.G. and A.C.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación, MICIU/AEI/10.13039/501100011033, under grant number PID2019-104331RB-I00.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author [A.C.-B.] upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chemical structure of uric acid, urate, 1,3-dimethyluric acid, and 9 methylxanthines tested as inhibitors of urate salts’ crystallization.
Figure 1. Chemical structure of uric acid, urate, 1,3-dimethyluric acid, and 9 methylxanthines tested as inhibitors of urate salts’ crystallization.
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Crystals 14 00768 g002
Figure 2. Effect of different methylxanthines (0.3 mM) on the crystallization time, expressed as the mean of three replicates ± SD of ∆ti of ammonium urate (NH4U), monosodium urate (MSUM), and potassium urate (KU) in artificial urine at 37 °C and pH 7.45 relative to controls (no methylxanthines). Table 1 shows the additional experimental conditions.
Figure 2. Effect of different methylxanthines (0.3 mM) on the crystallization time, expressed as the mean of three replicates ± SD of ∆ti of ammonium urate (NH4U), monosodium urate (MSUM), and potassium urate (KU) in artificial urine at 37 °C and pH 7.45 relative to controls (no methylxanthines). Table 1 shows the additional experimental conditions.
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Crystals 14 00768 g003
Figure 3. Effect of 3-methylxanthine and 7-methylxanthine concentration on the increase in crystallization time (∆ti, expressed as the mean of three replicates ± SD) of ammonium urate, monosodium urate, and potassium urate in artificial urine at 37 °C and pH 7.45 relative to controls (no methylxanthines). Table 1 shows the additional experimental conditions.
Figure 3. Effect of 3-methylxanthine and 7-methylxanthine concentration on the increase in crystallization time (∆ti, expressed as the mean of three replicates ± SD) of ammonium urate, monosodium urate, and potassium urate in artificial urine at 37 °C and pH 7.45 relative to controls (no methylxanthines). Table 1 shows the additional experimental conditions.
Crystals 14 00768 g003
Crystals 14 00768 g004
Figure 4. Combined effect of 3-methylxanthine and 7-methylxanthine concentrations on the increase in crystallization time (∆ti, expressed as the mean of three replicates ± SD) of ammonium urate (A) and potassium urate (B) at 37 °C and pH 7.45 relative to controls (no methylxanthines). Table 1 shows the additional experimental conditions.
Figure 4. Combined effect of 3-methylxanthine and 7-methylxanthine concentrations on the increase in crystallization time (∆ti, expressed as the mean of three replicates ± SD) of ammonium urate (A) and potassium urate (B) at 37 °C and pH 7.45 relative to controls (no methylxanthines). Table 1 shows the additional experimental conditions.
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Crystals 14 00768 g005
Figure 5. Effect of 7-methylxanthine concentration on crystallization of monosodium urate (initial uric acid concentration: 2.38 × 10−3 M [400 mg/L]) in conditions that mimicked synovial fluid (31 °C, pH 7.4, initial sodium concentration: 0.4 × 10−1 M, 96 h) expressed as mean ± SD (A) and image of a representative experiment at 96 h (B).
Figure 5. Effect of 7-methylxanthine concentration on crystallization of monosodium urate (initial uric acid concentration: 2.38 × 10−3 M [400 mg/L]) in conditions that mimicked synovial fluid (31 °C, pH 7.4, initial sodium concentration: 0.4 × 10−1 M, 96 h) expressed as mean ± SD (A) and image of a representative experiment at 96 h (B).
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Crystals 14 00768 g006
Figure 6. Effect of initial uric acid concentration on the crystallization of monosodium urate without (1) and with 50 µM of 7-methylxanthine (2) in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial sodium concentration: 0.4 × 10−1 M, 31 °C, pH: 7.4, incubation time: 96 h.
Figure 6. Effect of initial uric acid concentration on the crystallization of monosodium urate without (1) and with 50 µM of 7-methylxanthine (2) in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial sodium concentration: 0.4 × 10−1 M, 31 °C, pH: 7.4, incubation time: 96 h.
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Crystals 14 00768 g007
Figure 7. Effect of sodium and 7-methylxanthine concentrations on crystallization of monosodium urate in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial uric acid concentration: 400 mg/L, 31 °C, pH 7.4, incubation time: 96 h.
Figure 7. Effect of sodium and 7-methylxanthine concentrations on crystallization of monosodium urate in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial uric acid concentration: 400 mg/L, 31 °C, pH 7.4, incubation time: 96 h.
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Crystals 14 00768 g008
Figure 8. Effect of 7-methylxanthine concentration without (1) and with 96 mg/L calcium (2) on crystallization of monosodium urate in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial uric acid concentration: 400 mg/L, 31 °C, pH 7.4, incubation time: 96 h.
Figure 8. Effect of 7-methylxanthine concentration without (1) and with 96 mg/L calcium (2) on crystallization of monosodium urate in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial uric acid concentration: 400 mg/L, 31 °C, pH 7.4, incubation time: 96 h.
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Crystals 14 00768 g009
Figure 9. Effect of 7-methylxanthine concentration on crystallization of monosodium urate with no additions, addition of albumin (10 g/L), and addition of two different hyaluronic acid products (3 g/L each) in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial uric acid concentration: 400 mg/L, 31 °C, pH 7.4, incubation time: 96 h.
Figure 9. Effect of 7-methylxanthine concentration on crystallization of monosodium urate with no additions, addition of albumin (10 g/L), and addition of two different hyaluronic acid products (3 g/L each) in conditions that mimicked synovial fluid, expressed as the mean of three replicates ± SD. Initial uric acid concentration: 400 mg/L, 31 °C, pH 7.4, incubation time: 96 h.
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Crystals 14 00768 g010
Figure 10. Effect of uric acid concentration on monosodium urate crystallization in human plasma without 7-methylxanthine (1), with 50 µM of 7-methylxanthine (2), and with 100 µM of 7-methylxanthine; (3) 31 °C, incubation time: 96 h.
Figure 10. Effect of uric acid concentration on monosodium urate crystallization in human plasma without 7-methylxanthine (1), with 50 µM of 7-methylxanthine (2), and with 100 µM of 7-methylxanthine; (3) 31 °C, incubation time: 96 h.
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Crystals 14 00768 g011
Figure 11. SEM images of ammonium urate crystals from the kinetic–turbidimetric assays in artificial urine without methylxanthines (A), with 0.3 × 10−3 M 3-methylxanthine (B), and with 0.3 × 10−3 M 7-methylxanthine (C). Initial uric acid concentration: 2.97 × 10−3 M [500 mg/L], ammonium concentration: 2.64 × 10−1 M, pH 7.45.
Figure 11. SEM images of ammonium urate crystals from the kinetic–turbidimetric assays in artificial urine without methylxanthines (A), with 0.3 × 10−3 M 3-methylxanthine (B), and with 0.3 × 10−3 M 7-methylxanthine (C). Initial uric acid concentration: 2.97 × 10−3 M [500 mg/L], ammonium concentration: 2.64 × 10−1 M, pH 7.45.
Crystals 14 00768 g011
Crystals 14 00768 g012
Figure 12. SEM images of monosodium urate crystals from the kinetic–turbidimetric assays in artificial urine without methylxanthines (A), with 0.3 × 10−3 M 3-methylxanthine (B), and with 0.3 × 10−3 M 7-methylxanthine (C). Initial uric acid concentration: 2.97 × 10−3 M [500 mg/L], sodium concentration: 6.18 × 10−1 M, pH 7.45.
Figure 12. SEM images of monosodium urate crystals from the kinetic–turbidimetric assays in artificial urine without methylxanthines (A), with 0.3 × 10−3 M 3-methylxanthine (B), and with 0.3 × 10−3 M 7-methylxanthine (C). Initial uric acid concentration: 2.97 × 10−3 M [500 mg/L], sodium concentration: 6.18 × 10−1 M, pH 7.45.
Crystals 14 00768 g012
Crystals 14 00768 g013
Figure 13. SEM images of potassium urate crystals from the kinetic–turbidimetric assays in artificial urine without methylxanthines (A), with 0.3 × 10−3 M 3-methylxanthine (B), and with 0.3 × 10−3 M 7-methylxanthine (C). Initial uric acid concentration: 4.46 × 10−3 M (750 mg/L), potassium concentration: 6 × 10−1 M, pH 7.45.
Figure 13. SEM images of potassium urate crystals from the kinetic–turbidimetric assays in artificial urine without methylxanthines (A), with 0.3 × 10−3 M 3-methylxanthine (B), and with 0.3 × 10−3 M 7-methylxanthine (C). Initial uric acid concentration: 4.46 × 10−3 M (750 mg/L), potassium concentration: 6 × 10−1 M, pH 7.45.
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Table 1. Composition of each stock solution and final concentration in the synthetic urine solution. Synthetic urine samples were obtained by 1/4 dilution of a stock solution.
Table 1. Composition of each stock solution and final concentration in the synthetic urine solution. Synthetic urine samples were obtained by 1/4 dilution of a stock solution.
Ammonium Urate CrystallizationSodium Urate CrystallizationPotassium Urate Crystallization
Stock SolutionFinal ConcentrationStock SolutionFinal ConcentrationStock SolutionFinal Concentration
(g/L)(mM)(g/L)(mM)(g/L)(mM)
Na2SO4 · 10H2O12.59.712.59.712.59.7
NaH2PO4 · 2H2O4.87.74.87.74.87.7
Na2HPO4 · 12H2O11.27.811.27.811.27.8
NH4Cl56.52649.343.49.343.4
KCl24.381.324.381.3179600
NaCl26.1111.5134.557526.1111.5
Table 2. Summary of conditions and results for urate salts’ crystallization in synthetic urine.
Table 2. Summary of conditions and results for urate salts’ crystallization in synthetic urine.
Urate SaltI (M)[U−] (M)[Cation] (M)Ksp (M2) [19]SSti (min)Δti (min)
3-MX (0.3 mM)		Δti (min)
7-MX (0.3 mM)
NH4U0.523 × 10−30.2640.36 × 10−51114.69.36.7
NaU0.763 × 10−30.6182.79 × 10−5395.74.440
KU0.824.5 × 10−30.6009.63 × 10−5148.06.47.25
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Dietrich, J.; Grases, F.; Costa-Bauza, A. Effect of Methylxanthines on Urate Crystallization: In Vitro Models of Gout and Renal Calculi. Crystals 2024, 14, 768. https://doi.org/10.3390/cryst14090768

AMA Style

Dietrich J, Grases F, Costa-Bauza A. Effect of Methylxanthines on Urate Crystallization: In Vitro Models of Gout and Renal Calculi. Crystals. 2024; 14(9):768. https://doi.org/10.3390/cryst14090768

Chicago/Turabian Style

Dietrich, Jaume, Felix Grases, and Antonia Costa-Bauza. 2024. "Effect of Methylxanthines on Urate Crystallization: In Vitro Models of Gout and Renal Calculi" Crystals 14, no. 9: 768. https://doi.org/10.3390/cryst14090768

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