Adrian Rodriguez

Theobromine inhibits uric acid crystallization. A potential application in the treatment of uric acid nephrolithiasis.

Purpose To assess the capacity of methylxanthines (caffeine, theophylline, theobromine and paraxanthine) to inhibit uric acid crystallization, and to evaluate their potential application in the treatment of uric acid nephrolithiasis. Materials and Methods The ability of methylxathines to inhibit uric acid nucleation was assayed turbidimetrically. Crystal morphology and its modification due to the effect of theobromine were evaluated by scanning electron microscopy (SEM). The ability of theobromine to inhibit uric acid crystal growth on calculi fragments resulting from extracorporeal shock wave lithotripsy (ESWL) was evaluated using a flow system. Results The turbidimetric assay showed that among the studied methylxanthines, theobromine could markedly inhibit uric acid nucleation. SEM images showed that the presence of theobromine resulted in thinner uric acid crystals. Furthermore, in a flow system theobromine blocked the regrowth of post-ESWL uric acid calculi fragments. Conclusions Theobromine, a natural dimethylxanthine present in high amounts in cocoa, acts as an inhibitor of nucleation and crystal growth of uric acid. Therefore, theobromine may be clinically useful in the treatment of uric acid nephrolithiasis.

Efficacy of Mixtures of Magnesium, Citrate and Phytate as Calcium Oxalate Crystallization Inhibitors in Urine

PURPOSE The main aim of the current study was to evaluate the effectiveness of mixtures of magnesium, citrate and phytate as calcium oxalate crystallization inhibitors. MATERIALS AND METHODS A turbidimetric assay in synthetic urine was performed to obtain induction times for calcium oxalate crystallization in the absence and presence of different mixtures of inhibitors. The morphology of calcium oxalate crystals in the absence or presence of inhibitors and mixtures of the inhibitors was evaluated in 2 crystallization experiments at low and high calcium oxalate supersaturation. The crystals formed were examined using scanning electron microscopy. RESULTS Examination of crystallization induction times revealed clear inhibitory effects of magnesium, citrate and phytate on calcium oxalate crystallization, supporting usefulness in the treatment and prevention of calcium oxalate nephrolithiasis. Significant synergistic effects between magnesium and phytate were observed. Scanning electron microscopy images revealed that phytate is a powerful crystal growth inhibitor of calcium oxalate, totally preventing the formation of trihydrate and monohydrate. In addition to crystallization inhibition capacity, citrate and magnesium avoided calcium oxalate crystallization by decreasing its supersaturation. CONCLUSIONS The synergistic effect between magnesium and phytate on calcium oxalate crystallization suggests that a combination of these 2 compounds may be highly useful as antilithiasis therapy.

HPLC method for urinary theobromine determination: Effect of consumption of cocoa products on theobromine urinary excretion in children

OBJECTIVE To validate a simple method of urinary theobromine determination, to assess urinary theobromine levels in 80 healthy children and to relate these levels to consumption of cocoa products. DESIGN AND METHODS Urine samples were diluted, directly injected into an HPLC system, separated by gradient elution on a C18 column, and detected by UV spectrometry. The method was validated for linearity, limits of detection and quantification, imprecision, accuracy, recovery and interferences. The proposed method was used to assess 12-h day and 12-h night urinary theobromine excretion by 80 healthy children, divided into four groups based on consumption of cocoa products. In addition, urinary excretion of magnesium and oxalate, also present in cocoa, was measured in these four groups. RESULTS The method was linear to a theobromine concentration of 278μmol/L (50mg/L). LOD and LOQ for urine samples, diluted 1:5 (vol/vol) with water, were 1.1 and 3.6μmol/L respectively. Within-run and between-run imprecisions (CV) were each <2%. Average recovery was 99%, and analysis of a certified reference sample showed an error <2.5%. Theobromine excretion levels were significantly higher in healthy children with higher consumption of cocoa products (p<0.001), but oxalate (p=0.098) and magnesium (p=0.068) excretion levels did not differ significantly. CONCLUSION This validated method resulted in urinary theobromine determination with 100% recovery, without sample pretreatment. Urinary theobromine levels in healthy children were directly related to their consumption of cocoa products.

A new device for simple and accurate urinary pH testing by the Stone-former patient

PurposeUrinary pH is an important factor linked to renal stone disease and a useful marker in the treatment of urolithiasis. Although the gold standard for measuring urinary pH utilizes a glass electrode and a pH meter, at present dipstick testing is largely used to estimate urinary pH. However, the accuracy and precision of this method may be insufficient for making clinical decisions in patients with lithiasis. The aim of this study is to describe a new device for urinary pH testing.MethodsThe device includes a pH sensor based on differential measurement of an ISFET-REFET pair. The drawbacks associated with this type of configuration, namely short lifetime and manual fabrication, have been overcome in the prototype. An automatic one point calibration is performed when turning on the system. Two buffer solutions were utilized to determine the intra- and inter-day precision of the device. The pH of 30 fresh human urine samples was measured using a pH-meter, a dipstick and the new electronic device.ResultsIn some cases, dipstick measurements differed from those of the pH meter by more than 0.40 units, a clinically relevant discrepancy, whereas none of the measurements made with the new electronic device differed from the results of the pH-meter by more than 0.1 pH units.ConclusionsThis new electronic device has the possibility to be used by stone-formers to control their urinary pH at home, increasing the tools available for stone prevention and prophylaxis.

Exploration of Noncoding Sequences in Metagenomes

Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C) content, Codon Usage (Cd), Trinucleotide Usage (Tn), and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS) in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

Novel Colorimetric Determination of Phytate in Urine

ABSTRACT Phytate is a natural substance present in urine and biological fluids that is associated with health benefits. Existing methods for the determination of phytate are not applicable to routine measurements in urine by clinical laboratories. This study describes a new method for measuring urinary phytate. Following its purification and preconcentration from urine by solid-phase extraction, phytate may be determined colorimetrically using Al(III)-lumogallion or Al(III)-pyrocatechol violet. Following preconcentration, the Al(III)-lumogallion assay was linear for 0 to 15 µM phytate with a limit of detection of 0.183 µM. In contrast, the Al(III)-pyrocatechol violet system was linear with phytate concentration from 0 to 10 µM and provided a limit of detection of 0.010 µM with a preconcentration procedure. Both methods are simple, rapid, accurate, reliable, and sensitive. Comparisons using synthetic and human urine samples with a reference Al(III)-xylenol orange method showed that the Al(III)-pyrocatechol violet assay is a good alternative for measuring urinary phytate.

Orbitrap™ high-resolution mass spectrometry for the identification of amoxicillin crystalluria

Amoxicillin is semisynthetic penicillin that is widely prescribed to adult and pediatric patients. The use of amoxicillin can lead to crystalluria; however, the incidence of this adverse effect is unknown [1]. Light microscopy is insufficient for the complete characterization of these crystals. Over the past 20 years, clinicians have traditionally used Fourier transform infrared (FTIR) spectroscopy for the definitive identification of urinary crystals [2]. This method may be used in conjunction with other analytical techniques such as scanning electron microscopy (SEM) [3], nuclear magnetic resonance spectroscopy [4] and liquid (LC) or gas chromatography (GC) coupled with mass spectrometry (MS) [5, 6]. The aim of the present study was to assess an alternative method, OrbitrapTM high-resolution mass spectrometry (HRMS), for the identification of amoxicillin crystalluria. This method is a more rapid alternative to the combination of methods traditionally used by clinicians. A 49-year-old male was transferred to the emergency department of a secondary referral hospital in October 2017, with a diagnostic impression of stroke in the context of a hypertensive crisis. The patient’s medical history indicated that he was an active tobacco user, a heavy consumer of alcohol and a polydrug user. He had mild liver disease due to a hepatitis C viral infection and arterial hypertension. A left temporal arteriovenous malformation had been detected during a previous migraine study. The patient’s only home medication was methadone. Upon arrival at the first hospital (day 0), the patient was unconscious (Glasgow Coma Scale 6), and his blood pressure was 186/89 mmHg. An urgent cranial computed tomography (CT) scan revealed an extensive left temporoparietal intraparenchymal hematoma, and the patient was admitted to the intensive care unit. Orotracheal intubation and connection to mechanical ventilation were rapidly implemented. A urine drug screen (immunoassay) was positive for cocaine, opiates (administered therapeutically soon after admission) and methadone. Urinalysis showed a pH of 8.0 but no other abnormal results. Following consultation with the neurosurgery department of our tertiary referral hospital, the patient was transferred for surgery. During the first 12 h after admission, evacuation of the large parenchymal hematoma was performed. Empirical amoxicillin/clavulanic acid therapy (2 g every 8 h) was initiated due to a high clinical suspicion of bronchoaspiration, which was maintained for 5 days. Microbiological cultures of the patient’s urine and blood were negative; however, Escherichia coli (>106 colony-forming units/mL) was isolated from the bronchial aspirate. At 24 h postadmission (day 1), urinalysis was performed again as part of the routine examination. Direct examination of the urine on arrival at the emergency laboratory showed that it was very cloudy and granular (Figure 1). A dipstick test, performed at 8:00 a.m., indicated that the pH was 6.5 (confirmed by a pH meter), specific gravity was 1.030, protein level was 50 mg/dL, hemoglobin was 60 erythrocytes/μL and the reading for leukocyte esterase was negative. Following centrifugation and resuspension of the sediment, the specimen had a whitish and calcareous appearance. Optical *Corresponding author: Bernardino Barceló, Clinical Toxicology Unit, Clinical Analysis Department, Hospital Universitari Son Espases, Research Institute of Health Sciences (IdISBa), Palma de Mallorca, Spain, Phone: +87120627, Fax: +871909706, E-mail: bernardino.barcelo@ssib.es Adrian Rodriguez, Antonia Costa-Bauza and Félix Grases: Laboratory of Renal Lithiasis Research, University Institute of Health Sciences Research (IUNICS-IdISBa), University of Balearic Islands, Palma de Mallorca, Spain Marta Ocon Lopez: Department of Intensive Care Medicine, Hospital Universitari Son Espases, Palma de Mallorca, Spain Isabel Gomila: Clinical Analysis Department, Hospital Universitari Son Llàtzer, Research Institute of Health Sciences (IdISBa), Palma de Mallorca, Spain Maria Blanca Badal Cogul: Clinical Analysis Department, Hospital Universitari Son Espases, Palma de Mallorca, Spain

Factors Associated With the Lower Prevalence of Nephrolithiasis in Children Compared With Adults.

OBJECTIVE To determine the reasons behind the lower prevalence of kidney stones in children by assessing urinary lithogenic parameters in healthy children, healthy adults, and 3 groups of stone-former patients. METHODS The study subjects included 75 healthy adults, 105 healthy children, 62 patients with previous calcium oxalate monohydrate papillary stones, 120 patients with previous calcium oxalate monohydrate unattached stones, and 248 patients with previous calcium oxalate dihydrate stones. Twenty-four-hour urine samples were collected, and the urinary lithogenic parameters were measured. RESULTS Calcium, magnesium, and phosphorous concentration differed significantly between healthy children and adults. Except citrate, all solute/creatinine ratios differed between healthy children and adults. However, these differences were much more important in the cases of calcium and magnesium. The calcium/creatinine ratio was 2-fold lower, whereas the magnesium/creatinine ratio was 2-fold higher, in healthy children than that in healthy adults (P <.001 each). The calcium/creatinine ratio was higher and the citrate/creatinine ratio lower in calcium oxalate dihydrate stone formers than that in healthy adults. CONCLUSION Ratios of calcium and magnesium to creatinine, as well as morphoanatomic factors and lifestyle habits, may explain the lower prevalence of nephrolithiasis in children than those in adults.

Application of nuclear magnetic resonance spectroscopy for identification of ciprofloxacin crystalluria.

This is a report describing a previously healthy young patient, who experienced crystalluria and non-cholestatic acute liver injury after a single intravenous dose of 400mg. The nuclear magnetic resonance spectra confirmed that the urinary sediment in our patient was formed by pure ciprofloxacin. The nuclear magnetic resonance spectra ((1)H NMR) of the urine sediment are a good test to confirm the composition of the crystals observed by electron microscopy and infrared spectrum. The findings indicate the importance of adequate hydration, urinalysis, measurement of pH and liver enzyme levels, prior to treatment with ciprofloxacin. Our findings also indicate that ciprofloxacin should not be administered to patients with renal tubular acidosis, due to their high urinary pH.

Xanthine urolithiasis: Inhibitors of xanthine crystallization

Objective To identify in vitro inhibitors of xanthine crystallization that have potential for inhibiting the formation of xanthine crystals in urine and preventing the development of the renal calculi in patients with xanthinuria. Methods The formation of xanthine crystals in synthetic urine and the effects of 10 potential crystallization inhibitors were assessed using a kinetic turbidimetric system with a photometer. The maximum concentration tested for each compound was: 20 mg/L for 3-methylxanthine (3-MX); 40 mg/L for 7-methylxanthine (7-MX), 1-methylxanthine (1-MX), theobromine (TB), theophylline, paraxanthine, and caffeine; 45 mg/L for 1-methyluric acid; 80 mg/L for 1,3-dimethyluric acid; and 200 mg/L for hypoxanthine. Scanning electron microscopy was used to examine the morphology of the crystals formed when inhibitory effects were observed. Results Only 7-MX, 3-MX, and 1-MX significantly inhibited xanthine crystallization at the tested concentrations. Mixtures of inhibitors had an additive effect rather than a synergistic effect on crystallization. Conclusion Two of the inhibitors identified here—7-MX and 3-MX—are major metabolites of TB. In particular, after TB consumption, 20% is excreted in the urine as TB, 21.5% as 3-MX, and 36% as 7-MX. Thus, consumption of theobromine could protect patients with xanthinuria from the development of renal xanthine calculi. Clinical trials are necessary to demonstrate these effects in vivo.