Isabel Gomila

Phytotherapy and renal stones: the role of antioxidants. A pilot study in Wistar rats

Since ancient times, various herbal preparations have been used in renal lithiasis therapy, but conclusive scientific data on their therapeutic effects and efficacy are not available. To address this issue, the present study evaluated the antilithiasic activity of a traditional Mallorcan herbal preparation, and compared its effects with those of the antioxidant flavonoids, catechin and epicatechin. Thirty-six male Wistar rats were assigned randomly to four groups (nxa0=xa09): a control group, a catechin (CAT) treatment group, an epicatechin (EPI) treatment group, and a group treated with a folk herbal extract (FHE). After 16xa0days of treatment, calcium oxalate lithiasis was induced in the rats using ethylene glycol. After 8xa0days (treatmentxa0+xa0ethylene glycol), 24-h rat urine was collected, the animals were sacrificed and their kidneys were removed for histological and chemical analysis. The calcium concentration in kidney tissue was significantly lower in the CAT-treated (2.4xa0±xa00.3xa0mg/g), EPI-treated (1.8xa0±xa00.3xa0mg/g) and FHE-treated (2.1xa0±xa00.3xa0mg/g) groups, than in the control group (5.4xa0±xa01.4xa0mg/g). Examination of paraffin-embedded kidney sections showed that control group rats had the greatest amount of calcification. There were no significant differences between control and treated groups with respect to urinary calcium, magnesium, oxalate and citrate concentrations. These results demonstrate the ability of herbal preparations and antioxidants to prevent the development of papillary and intratubular calcification in the kidney.

Urinary pH and renal lithiasis

Formation of calcium oxalate crystals, either as monohydrate or dihydrate, is apparently unrelated to urinary pH because the solubilities of these salts are practically unaltered at physiologic urinary pH values. However, a urinary pH <5.5 or >6.0 may induce uric acid or calcium phosphate crystals formation, respectively, which under appropriate conditions may induce the development of the calcium oxalate calculi. We assessed the relationship between the urinary pH and the formation of different types of calculi. A retrospective study in 1,478 patients was done. We determined the composition, macrostructure, and microstructure of the calculi and the urinary pH, 50.9% of calcium oxalate monohydrate unattached calculi were present in patients with urinary pH <5.5. We found that 34.1 and 41.5% of calcium oxalate dihydrate calculi were present in patients with urinary pH <5.5 and >6.0, respectively. Infectious calculi were found primarily in patients with urinary pH >6.0 (50.7%). Only calcium oxalate monohydrate papillary calculi were associated with urinary pH between 5.5 and 6.0 (43.1%). Urine of pH <5.5 shows an increased capacity to develop uric acid crystals, which can act as a heterogeneous nuclei of calcium oxalate crystals. In contrast, urine of pH >6.0 has an increased capacity to develop calcium phosphate crystals, which can act as a heterogeneous nuclei of calcium oxalate crystals. Oxalate monohydrate papillary calculi were associated to pH between 5.5 and 6.0 because the injured papilla acts as a heterogeneous nucleant. Consequently, measurement of urinary pH may be used to evaluate the lithogen risk of given urine.

Phytotherapy in a rat model of hyperoxaluria: the antioxidant effects of quercetin involve serum paraoxonase 1 activation

Serum paraoxonase 1 (PON1) has been reported to be an important contributor to the antioxidant and anti-inflammatory activities of HDL, avoiding LDL oxidation. The activity of this enzyme is reduced in patients with renal insufficiency, caused by elevated oxidative stress and disturbances of apolipoprotein metabolism. Therapeutic utilization of antioxidants to control renal oxidative stress may be an effective therapy in renal protection. The aim was to investigate the protective effects of several antioxidant compounds against the oxidative stress associated to renal failure induced by ethylene glycol (EG), focusing on the possible role of serum PON1 activity. Fifty-four male Wistar rats were randomly assigned to six groups (n = 9): an untreated control (C) group, an EG-treated group, a catechin (CAT)-treated group, an epicatechin (EPI)-treated group, a quercetin (QUE)-treated group and a folk herbal extract (FHE)-treated group. After 16 d of treatment, calcium oxalate lithiasis was induced in the rats using EG. After eight days (treatment + EG), the animals were sacrificed. EG treatment impaired kidney composition, increased oxidative damage, and decreased serum paraoxonase and arylesterase activities. CAT, QUE and the FHE Fagolitos improved oxidative status by enhancing antioxidant defenses – superoxide dismutase and PON1 activities – and reducing oxidative damage, thus reinforcing the idea of a possible role of PON1 in the protective effects of QUE against the deleterious consequences of oxidative stress in kidney.

A simple and rapid colorimetric method for determination of phytate in urine

Phytate is a natural product present in urine and biological fluids that is associated with health benefits, such as the prevention of calcium renal stone formation. The available methods for phytate analysis in urine all require elaborate instrumentation and cannot be routinely applied in clinical laboratories. Here, we describe a simple procedure for urinary phytate determination, employing colorimetric detection. Our method requires purification and preconcentration of phytate via solid-phase extraction prior to colorimetric detection employing Fe(III)–thiocyanate. The working linear range of the assay is 0–5xa0μM phytate. The limit of detection is 0.055xa0μM. The relative standard deviation obtained upon assay of samples containing 2xa0μM phytate was 3.5xa0%. Several urine samples were analyzed using an alternative method based on the detection of phosphorus; the results of the two assays were comparable. Our novel method of phytate analysis in human urine is simple, rapid (3xa0h for 10 samples), accurate, precise, reliable, and highly sensitive. The assay can be run in most analytical laboratories and does not require sophisticated instrumentation.

Urinary lithogenesis risk tests: Comparison of a commercial kit and a laboratory prototype test

Abstract Objective. Renal stone formation is a multifactorial process depending in part on urine composition. Other parameters relate to structural or pathological features of the kidney. To date, routine laboratory estimation of urolithiasis risk has been based on determination of urinary composition. This process requires collection of at least two 24 h urine samples, which is tedious for patients. The most important feature of urinary lithogenic risk is the balance between various urinary parameters, although unknown factors may be involved. The objective of this study was to compare data obtained using a commercial kit with those of a laboratory prototype, using a multicentre approach, to validate the utility of these methods in routine clinical practice. Material and methods. A simple new commercial test (NefroPlus; Sarstedt AG & Co., Nümbrecht, Germany) evaluating the capacity of urine to crystallize calcium salts, and thus permitting detection of patients at risk for stone development, was compared with a prototype test previously described by this group. Urine of 64 volunteers produced during the night was used in these comparisons. The commercial test was also used to evaluate urine samples of 83 subjects in one of three hospitals. Results. Both methods were essentially in complete agreement (98%) with respect to test results. The multicentre data were: sensitivity 94.7%; specificity 76.9%; positive predictive value (lithogenic urine) 90.0%; negative predictive value (non-lithogenic urine) 87.0%; test efficacy 89.2%. Conclusion. The new commercial NefroPlus test offers fast and cheap evaluation of the overall risk of development of urinary calcium-containing calculi.

Non-infectious phosphate renal calculi: Fine structure, chemical and phase composition

Abstract Background. Chemical composition of internally non-homogeneous phosphate stones should be related to the conditions prevailing during the formation of each individual part. Objective. The object of this paper was to provide a detailed study of phosphate stone composition on the micro- and macro-scales. Methods. Fine inner structure, chemical and phase composition of 10 phosphate calculi from different patients were determined by chemical (wet) analysis, observation by scanning microscope, semi-quantitative determination of Ca, Mg, P and C by energy dispersive X-ray and by X-ray diffraction. Results. Eight calculi are formed by amorphous calcium phosphate and two by hydroxyapatite. Magnesium was inversely related to Ca/P ratio. Point chemical composition of solid phase varies in wide limits, i.e. composition of calculus interior is highly inhomogeneous on the microscale. All studied calculi contained an abundance of organic matter incorporated in their volume; the content of carbon was double the calcium content in molar quantities. Conclusions. Phosphate renal calculi with the low Ca/P molar ratio predominantly consist of amorphous calcium phosphate whereas those with a high Ca/P molar ratio are composed of poorly crystalline hydroxyapatite which can be partially carbonated. Magnesium may be an inhibitor of HAP formation from urine. Abundant organic matter incorporated into the calculus volume indicates its decisive role at stone formation. Variable point composition of stones implies widely varying conditions during their development.

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

Value of glycolic acid analysis in ethylene glycol poisoning: A clinical case report and systematic review of the literature

OBJECTIVEnTo evaluate the clinical utility of glycolic acid (GA) determination in the diagnosis and prognosis of ethylene glycol (EG) intoxications.nnnMETHODnSystematic review of serum and/or urine GA concentrations available in the literature in cases of EG poisoning. Present a clinical case in which the determination of the GA was decisive.nnnRESULTSnIn total, 137 patients were included. Serum GA concentrations (but not EG) of patients who survive are different from those who die. The optimal cut-off of serum GA to predict mortality was 990.5mg/L (sensitivity 85.2%, specificity 54.3%) with an Odds Ratio of 6.838 (2.868-16.302). In our clinical case, serum EG was negative; however, urine GA was positive (1230.7mg/L).nnnCONCLUSIONSnIn all suspected cases of EG poisoning, it is advisable to carry out the simultaneous analysis of EG and GA.

Building Bridges between Clinical and Forensic Toxicology Laboratories

BACKGROUNDnClinical and forensic toxicology can be defined as two disciplines involving the detection, identification and measurement of xenobiotics in biological and non-biological samples to assist in the diagnosis, treatment, prognosis and prevention of poisonings and to disclose causes and contributory causes of fatal intoxications, respectively.nnnOBJECTIVEnThis article explores the close connections between clinical and forensic toxicology in overlapping areas of interest.nnnMETHODSnAn update has been carried out of the following seven areas of interest in analytical toxicology: doping control, Sudden Cardiac Death (SCD), brain death, Sudden Infant Death Syndrome (SIDS) and Munchausen Syndrome by Proxy (MSBP), prenatal exposure to drugs and Fetal Alcohol Syndrome (FAS), Drug-Facilitated Crimes (DFC) and intoxications by new psychoactive substances (NPS).nnnRESULTSnWhile issues such as SCD, SIDS or doping control are investigated mainly in forensic laboratories, others such as prenatal exposure to drugs or FAS are mainly treated in clinical laboratories. On the other hand, areas such MSBP, DFC or the intoxications by NPS are of interest in both laboratories. Some of these topics are initially treated in hospital emergency departments, involving clinical laboratories and sometimes lately derived to forensic laboratories. Conversely, cases with initial medicallegal implications and fatalities are directly handled by forensic toxicology, but may trigger further studies in the clinical setting.nnnCONCLUSIONnMany areas of common interest between clinical and forensic laboratories are building bridges between them. The increasing relationships are improving the growth, the reliability and the robustness of both kinds of laboratories.

2,4-Diamino-N10-methylpteroic acid (DAMPA) crystalluria in a patient with osteosarcoma treated with carboxypeptidase-G2 rescue after high-dose methotrexate-induced nephrotoxicity

BACKGROUNDnHigh-dose methotrexate (HDMTX) therapy is a key component of many chemotherapy protocols. However, some patients develop HDMTX-induced nephrotoxicity. Carboxypeptidase-G2 (CPDG2) hydrolyses MTX into 2,4-diamino-N10-methylpteroic acid (DAMPA) and glutamic acid, and is used as a rescue agent in patients with nephrotoxicity and delayed elimination. Despite the frequency of HDMTX-induced renal injury, crystalluria is uncommon. Furthermore, crystals are rarely identified by conventional chemical methods.nnnOBJECTIVEnTo determine the composition of crystalluria in a patient with osteosarcoma who was treated with CPDG2.nnnMETHODSnCrystalluria was evaluated by optical microscopy, and chemical identification was performed by Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and Orbitrap™ high-resolution mass spectrometry (HRMS).nnnRESULTSnThe HRMS spectra of the patients urine sediment showed a main peak at m/z 326.13, corresponding to the molecular mass of DAMPA [(C15H15O2N7)u202f+u202fH+]. The FT-IR spectral patterns of the sediment and DAMPA were not identical. SEM was unable to identify the crystal.nnnCONCLUSIONnDAMPA crystalluria was identified by Orbitrap™ HRMS in a patient treated with CPDG2 after HDMTX nephrotoxicity. This case reinforces the need to implement adequate measures to prevent nephrotoxicity. In cases of HDMTX-induced nephrotoxicity, urine sediment analysis should be requested.