Effect of theobromine on dissolution of uric acid kidney stones

Abstract

Purpose
Uric acid renal lithiasis has a high prevalence and a high rate of recurrence. Removal of uric acid stones can be achieved by several surgical techniques (extracorporeal shock wave lithotripsy, endoscopy, laparoscopy, open surgery). These stones can also be eliminated by dissolution within the kidneys, because the solubility of uric acid is much greater when the pH is above 6. At present, N-acetylcysteine with a urinary basifying agent is the only treatment proposed to increase the dissolution of uric acid stones. In this paper, we compare the effect of theobromine and N-acetylcysteine on the in vitro dissolution of uric acid calculi in artificial urine at pH 6.5.

Methods
The dissolution of uric acid renal calculi was performed in a temperature-controlled (37 °C) chamber. A peristaltic pump was used to pass 750 mL of synthetic urine (pH 6.5) through a capsule every 24 h. Stone dissolution was evaluated by measuring the change in weight before and after each experiment.

Results
N-acetylcysteine increased the dissolution of uric acid calculi, but the effect was not statistically significant. Theobromine significantly increased the dissolution of uric acid calculi. Both substances together had the same effect as theobromine alone. The addition of theobromine to a basifying therapy that uses citrate and/or bicarbonate is a potential new strategy for the oral chemolysis of uric acid stones.

Conclusion
Theobromine may prevent the formation of new stones and increase the dissolution of existing stones.

Full text

Vol.:(0123456789)
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World Journal of Urology (2022) 40:2105–2111
https://doi.org/10.1007/s00345-022-04059-3
ORIGINAL ARTICLE
Effect oftheobromine ondissolution ofuric acid kidney stones
FrancescaJulià1· AntoniaCosta‑Bauza1 · FranciscoBerga1· FelixGrases1
Received: 25 February 2022 / Accepted: 16 May 2022 / Published online: 11 June 2022
© The Author(s) 2022
Abstract
Purpose Uric acid renal lithiasis has a high prevalence and a high rate of recurrence. Removal of uric acid stones can be
achieved by several surgical techniques (extracorporeal shock wave lithotripsy, endoscopy, laparoscopy, open surgery). These
stones can also be eliminated by dissolution within the kidneys, because the solubility of uric acid is much greater when
the pH is above 6. At present, N-acetylcysteine with a urinary basifying agent is the only treatment proposed to increase
the dissolution of uric acid stones. In this paper, we compare the effect of theobromine and N-acetylcysteine on the invitro
dissolution of uric acid calculi in artificial urine at pH 6.5.
Methods The dissolution of uric acid renal calculi was performed in a temperature-controlled (37°C) chamber. A peristaltic
pump was used to pass 750mL of synthetic urine (pH 6.5) through a capsule every 24h. Stone dissolution was evaluated
by measuring the change in weight before and after each experiment.
Results N-acetylcysteine increased the dissolution of uric acid calculi, but the effect was not statistically significant. Theobro-
mine significantly increased the dissolution of uric acid calculi. Both substances together had the same effect as theobromine
alone. The addition of theobromine to a basifying therapy that uses citrate and/or bicarbonate is a potential new strategy for
the oral chemolysis of uric acid stones.
Conclusion Theobromine may prevent the formation of new stones and increase the dissolution of existing stones.
Keywords Theobromine· N-acetylcysteine· Uric acid renal calculi· Dissolution
Introduction
Uric acid renal lithiasis has a high prevalence, in that it
accounts for more than 10% of kidney stones, and a high
rate of recurrence, in that an individual may form multiple
stones within a single year [1, 2]. The increasing prevalence
of obesity and metabolic syndrome may be responsible for
the significant increase in uric acid renal lithiasis during
recent years [3, 4]. Therefore, uric acid renal lithiasis is a
common, significant, and serious public health problem.
Uric acid renal lithiasis may be prevented by oral adminis-
tration of urinary alkalinizers, such as citrate, and/or inhibi-
tors of uric acid crystallization, such as theobromine (TB)
[5]. Removal of existing uric acid stones can be achieved
by several surgical techniques, including extracorporeal
shock wave lithotripsy, endoscopy, laparoscopy, and open
surgery. However, uric acid stones can also be eliminated
noninvasively by dissolution within the kidneys, because the
solubility of uric acid increases greatly at pH values above
6. Obviously, the dissolution rate depends on the size of
the stone and its location in the kidney, and greater irriga-
tion increases the rate of dissolution. Alkalinization of the
urine can be achieved with high doses of citrate, which is
sometimes accompanied by use of bicarbonate. However, in
some cases the oral consumption of high doses of citrate or
citrate and bicarbonate can lead to stomach discomfort, and
can induce the formation of sodium urate shells on the uric
acid stones if used for long periods [6]. Nevertheless, stone
dissolution without surgery has clear advantages, despite not
being widely used in clinical practice [7–10].
N-acetylcysteine (NAC) with a urinary basifying agent
has been proposed to increase the dissolution of uric acid
stones [11]. NAC is a mucolytic agent that acts by reducing
the viscosity of bronchial secretions. It works by cleaving
the disulfide bridges of mucoproteins, making them less
* Antonia Costa-Bauza
antonia.costa@uib.es
1 Laboratory ofRenal Lithiasis Research, University Institute
ofHealth Sciences Research (IUNICS-IdISBa), University
ofBalearic Islands, 07122PalmadeMallorca, Spain
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2106 World Journal of Urology (2022) 40:2105–2111
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viscous [12], although this combination is not recommended
in the current guidelines for uric acid stones.
In this paper we compared the effect of TB and NAC on
the invitro dissolution of uric acid stones in synthetic urine.
Materials andmethods
Reagents andsolutions
Uric acid, TB, NAC and escin were from Sigma-Aldrich
(St. Louis, MO, USA) and synthetic urine components
were from Panreac (Montcada i Reixac, Barcelona, Spain).
Chemicals of analytical reagent-grade purity were dissolved
in ultra-pure deionized water from a Milli-Q system. A uric
acid stock solution was prepared daily by dissolving 0.4g/L
uric acid with 1M NaOH (final pH: 10.52). A solution of
“concentrated” synthetic urine was prepared by dissolving
double the amounts of all substances listed in Table1. Cal-
cium and oxalate were not included to prevent crystallization
of calcium oxalate. The pH of this “concentrated” synthetic
urine was adjusted to 6.20. During experiments, equal vol-
umes of a uric acid solution and the “concentrated” synthetic
urine were mixed, so the final concentration of uric acid was
0.2g/L and the concentrations of other compounds were as
indicated in Table1.
Experimental procedure
Post-extracorporeal shock wave lithotripsy (ESWL) frag-
ments of uric acid stones were selected from a collection of
anonymous kidney stone samples from the Laboratory of
Renal Lithiasis Research belonging to University Institute
of Health Sciences Research of the University of Balearic
Islands. This collection has been generated from the rou-
tine kidney stone diagnostic study service that the Labora-
tory of Renal Lithiasis Research performs for the hospitals
of the Balearic Islands Community. Kidney stones were
studied and classified using the general protocol adopted
by our laboratory. This methodology includes the use of
optical stereomicroscopy, infrared spectrometry and scan-
ning electron microscopy (SEM) [13]. All selected frag-
ments had similar morphology and size.
In vitro dissolution of four uric acid calculi was per-
formed simultaneously in a temperature-controlled
chamber which remained at 37°C during the course of
experiments (48h). In each experiment, four hermetic flow
capsules (Fig.1) were used, each containing 1 fragment
of a uric acid calculus with no pre-treatment. A multi-
channel peristaltic pump was used to transfer the solution
of “concentrated” synthetic urine (solution A; pH 6.20),
with or without the experimental additive (see below) at a
rate of 375mL/day and a solution of 0.4g/L of uric acid
(solution B; pH 10.52) at the same rate. Both solutions
were maintained at 37°C and were mixed in a T connec-
tion before introduction into the capsule. Thus, 750mL of
synthetic urine (final pH: 6.5) passed through the capsule
every 24h. This is approximately the volume of urine that
typically passes through a single human kidney each day.
The effects of adding 40mg/L TB, 20 mg/L NAC,
40mg/L escin or a mixture of 20mg/L NAC with 40mg/L
TB (NAC + TB) on the dissolution of uric acid calculi
were compared with the results obtained from controls
treated with synthetic urine with no admixtures.
Experiments using a higher concentration of TB
(80mg/L) and an incubation period of 168h with 40mg/L
of TB were also performed.
Table 1 Composition of
synthetic urine
Final pH = 6.5
Substance Concentra-
tion (g/L)
Na2SO4·10H2O 3.12
MgSO4·7H2O 0.73
NH4Cl 2.32
KCl 6.07
NaH2PO4·2H2O 1.21
Na2HPO4·12H2O 2.80
NaCl 6.53
Uric acid 0.20 Fig. 1 Experimental model used to examine the effect of different
treatments on the dissolution of uric acid stones. See “Material and
methods” for a description
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2107World Journal of Urology (2022) 40:2105–2111
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Evaluation
The calculi were dried at 37°C for 24h before and after
each experiment until they reached constant weight, deter-
mined using a precision balance. Fragment dissolution was
calculated as the change in weight. Mean dissolution was
determined and standardized by calculating the relative mass
decrease, and thus did not consider the effect of surface area.
The morphological and structural characteristics of the
samples, before and after dissolution, were observed using
scanning electron microscopy (Hitachi S-3400N) coupled
with RX energy dispersive microanalysis (Bruker AXS
XFlash Detector 4010).
Statistics
The normality of data distributions was determined by
inspection of plots. Data were presented as means with 95%
confidence intervals (CIs). For continuous variables, 3 or
more groups were compared using ANOVA with the Bon-
ferroni post hoc correction, and 2 groups were compared
using Student’s t test. A two-tailed p value less than 0.05 was
considered statistically significant. Statistical analyses were
performed using SPSS version 25.0 (SPSS Inc., Chicago,
IL, USA).
Results
We first examined the effect of NAC, TB, and NAC + TB on
the dissolution of uric acid stones in artificial urine (Fig.2).
Relative to the control, NAC treatment increased dissolution,
although this effect was not statistically significant. TB treat-
ment significantly increased dissolution, and the effect was
similar for TB and NAC + TB. Thus, while treatment with
NAC was not statistically significant, treatments with TB
and NAC + TB were. Escin did not increase stone dissolu-
tion (data not shown). Notably, there was great variability
Fig. 2 Effect of TB (40mg/L),
NAC (20mg/L), and a mixture
of NAC (20mg/mL) + TB
(40mg/mL) on the dissolution
of uric acid stones at pH 6.5.
Percentage of dissolution was
expressed as mean ± 95% CI A
and as median ± interquartile
range B, with 10 replicates per
group. *Significantly different
from the Control
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2109World Journal of Urology (2022) 40:2105–2111
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among identically treated samples in these experiments
(Fig.2), presumably because of the different structures of
the calculi. Obviously, the most porous stones dissolved at
a higher rate than the most compact stones, but other factors
can also affect dissolution, such as organic matter coating
the stones (Fig.3 A,B). Our SEM images showed that NAC
facilitated the elimination of organic matter layers on the
stones (Fig.3 C,D) and that TB accelerated the dissolution
of the uric acid crystals (Fig.3 E,F). Despite their different
effects, we observed no additive or synergistic effects when
NAC and TB were used together.
When stones were incubated for 168h, we identified areas
with significant amounts of sodium and potassium urates
(Fig.3 G), such as those observed on the surface of some
uric acid kidney stones (Fig.3 H).
When we used a higher TB concentration (80mg/L), the
dissolution was greater than in the control (21.9 vs 17.1%),
but was not significantly different from that obtained with
the lower TB concentration.
Discussion
TB is an alkaloid molecule in the xanthine family that occurs
in the cocoa ‘bean’, and dark chocolate consists of about 1
to 4% TB [14]. TB is related to caffeine and theophylline,
but it has weaker effects on the central nervous system than
caffeine [15]. Due to the structural characteristics of TB,
it can inhibit uric acid crystallization, especially when the
urinary concentration is greater than 15mg/L [16]. Thus,
TB is the first potential inhibitor of uric acid crystalliza-
tion to be described. About 20% of ingested TB is excreted
in the urine [17, 18]. TB is currently accepted for use as
a diuretic and for its vasodilatory effects, and is therefore
used to treat patients with high blood pressure [19]. Previous
research indicated that saponins (such as ginseng extract),
glycosaminoglycans, and glycoproteins also hindered the
crystallization of uric acid [20]. However, the effects of these
substances were due to their alteration of the surface tension
of water; they are not typical crystallization inhibitors and
they do not affect nucleation or crystal growth, in which a
substance is adsorbed onto the faces of the crystal. Impor-
tantly, these substances also do not elicit dose–response
relationships.
Normally, a substance that inhibits the formation of
ionic crystals also inhibits crystal dissolution. For example,
phytate inhibits the crystallization of calcium salts (oxalate
and calcium phosphates), but also inhibits their dissolution
[21]. However, we showed here that TB, which was previ-
ously determined to inhibit crystallization, promoted crystal
dissolution (Fig.2). In fact, a recent study found that TB
and uric acid molecules interacted in solution, to form new
tetrameric cluster species [22]. Obviously, the formation of
these species would decrease the supersaturation of uric acid
and facilitate the dissolution of crystals. It is interesting to
note that the action of TB on uric acid stones was completely
different from that of NAC. Thus, NAC degraded deposits of
organic matter that covered the stone crystals (Fig.3 C,D),
but TB accelerated the dissolution of the crystals (Fig.3
E,F). Although each substance altered stone shape, TB
was more effective at stone dissolution and there were no
apparent additive or synergistic effects between these two
substances.
We also found that escin had no significant effect on the
dissolution of uric acid stones.
It is important to note that there was variability of the dis-
solution results obtained from the same treatment (Fig.2),
even though we used a stereoscopic microscope to select
stone fragments that were as similar as possible. Our SEM
results indicated this was likely because stones that appeared
macroscopically similar, had major differences in micro-
structure. In particular, the stones differed in the presence
of porosities, the distribution of organic matter and crystal
size. In fact, we observed these differences even within an
individual calculus. It is also likely that the position of the
fragment within the capsule and the flow of liquid around
the fragment influenced dissolution.
Our long term (168h) experiments indicated the presence
in controls of small areas of the stones in which there was
formation of deposits of sodium/potassium urate crystals
(Fig.3 G). Obviously, the formation of these deposits is a
consequence of the high pH and the high uric acid concen-
tration. Unfortunately, these deposits cannot dissolve at high
pH. In the presence of TB, as the supersaturation of the urate
salt decreases due to the formation of urate-TB clusters,
there is reduced formation of these precipitates. In long-term
dissolution processes, the formation of urate precipitates can
become significant when the pH is very high. In this case,
TB can prevent the formation of new stones and can also
reduce the formation of insoluble urates, by decreasing the
supersaturation of existing urates. In any case, a pH above
7 should be avoided, because this can lead to the forma-
tion of hard shells of sodium/potassium urate or apatite salts
that coat the stone, making dissolution impossible, and also
Fig. 3 SEM images of uric acid stones. A, B Before dissolution: sur-
face and higher magnification, showing compact uric acid crystals. C,
D After NAC treatment: surface, showing detachment of the exter-
nal layer of organic matter and higher magnification. E, F After TB
treatment: surface and higher magnification, showing partial dissolu-
tion of uric acid crystals. G Sodium and potassium urate crystals on
the surface of a uric acid stone after incubation in control solution for
168h. H Sodium and potassium urate needle-like crystals formed “in
vivo” on the surface of a uric acid stone
◂
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2110 World Journal of Urology (2022) 40:2105–2111
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because uric acid solubility does not significantly increase
above that pH value [23]. The formation of insoluble urates
(sodium/potassium) [6] will only take place when, due to
the concentrations of uric acid, sodium and/or potassium
in urine, they exceed the conditions of supersaturation of
these salts in this medium. Under conditions of low concen-
tration of uric acid, sodium and potassium in urine (very
diluted urine due to high water intake, and/or administra-
tion of allopurinol), this supersaturation will not be reached,
so this precipitation will not occur. The higher the urinary
pH, by increasing the ionization of uric acid [11], the urate
crystallization process will also be favored. Theobromine
binds to uric acid forming tetrameric species, increasing its
solubility [22], so to some extent, it can also prevent the
formation of insoluble urates. Fortunately, it seems that
the formation of these insoluble urates, which have been
observed in "in vitro" experiments [6], and which we have
also detected in the study presented in this paper, is not very
frequent, although we have detected their presence in kidney
stones in some patients (Fig.3 H).
Finally, our experiments also indicated that use of a
higher concentration of TB (80mg/L) provided no addi-
tional benefit.
Conclusions
When using oral chemolysis to treat uric acid renal stone
formers, the addition of an appropriate amount of TB to a
basifying therapy, consisting of citrate and/or bicarbonate
may improve outcome. TB appears to have two impor-
tant effects: it prevents the formation of new stones and it
increases the dissolution of existing stones. TB can also
minimize the formation of insoluble sodium/potassium urate
deposits. The U.S. Food and Drug Administration considers
TB to be ‘generally regarded as safe’. We therefore suggest
that the results presented here should be confirmed by clini-
cal trials.
Author contributions FJ: data collection, data analysis, manuscript
writing. AC-B: protocol development, data analysis, manuscript writ-
ing/editing. FB: protocol development, data collection. FG: protocol
development, data analysis, manuscript writing/editing.
Funding Open Access funding provided thanks to the CRUE-CSIC
agreement with Springer Nature. Grant PID2019-104331RB-I00
funded by MCIN/AEI/10.13039/501100011033.
Declarations
Conflict of interest The authors declare that they have no conflicts of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent Informed consent is not applicable in the study.
Open Access This article is licensed under a Creative Commons Attri-
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