Search Results (84)

Background: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency. Methods: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach’s applicability in putative biomarker discovery. Results: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness. Conclusions: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential. Full article

Background/Objectives: In diabetic ketoacidosis (DKA), absolute insulin deficiency and elevation of counter-regulatory hormones may cause osmotic diuresis and water and electrolyte loss, which may lead to dehydration and renal failure. Fluids with high Na content are preferred in the DKA fluid therapy algorithm due to the association of Na with β-Hydroxybutyrate (β-HB) and the renal excretion of Na-β-HB. However, these fluids may cause hyperchloremic metabolic acidosis due to their high chloride concentration. In the literature, base-excess chloride (BE_Cl) has been suggested as a better approach for assessing the effect of chloride on acid–base status. Our aim in this study was to investigate the effect of fluids with BE_Cl values less than zero versus those with values equal to or greater than zero on the metabolic acid–base status in the first 6 h of DKA. Methods: This retrospective study included DKA cases managed in the tertiary intensive care units of five hospitals in the last 10 years. Patients were divided into two groups according to the Na-Cl difference of the administered fluids during the first 6 h of treatment: Group I [GI, fluids with Na-Cl difference = 0, chloride-rich group] and Group II [GII, fluids with Na-Cl difference > 32 mmol, chloride non-rich group]. Demographic data, blood gas analysis results, types and amounts of administered fluids, urea–creatinine values, and urine ketone levels were recorded. Results: Thirty-five patients with DKA in the ICU were included in the study (GI; 22 patients, GII; 13 patients). There was no difference between the patients in the two groups in terms of age, gender, and LOS-ICU. According to the distribution of the administered fluids, the main fluid administered in GI was 0.9% NaCl, whereas in the GII, it was bicarbonate, Isolyte-S, and 0.9% NaCl. In GI, the chloride load administered was higher; the BE_Cl level of the fluids was lower than in GII. At the end of the first 6 h, although sodium and strong ion gap values were similar, patients in GI were more acidotic due to iatrogenic hyperchloremia and, as a result, were more hypocapnic than GII. Conclusions: In conclusion, administering chloride-rich fluids in DKA may help reduce unmeasured anion acidosis. Still, risks cause iatrogenic hyperchloremic acidosis, which can hinder the expected resolution of acidosis and increase respiratory workload. Therefore, it is suggested that DKA guidelines be revised to recommend an individualized approach that avoids chloride-rich fluids and includes monitoring of metabolic parameters like Cl and BE_Cl. Full article

Gitelman syndrome (GS) is a rare autosomal recessive renal tubulopathy characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria due to mutations in the SLC12A3 gene. This case report presents a 54-year-old African American female with near syncope and palpitations. The patient had a history of intermittent palpitations and generalized anxiety disorder and was previously diagnosed with GS. On presentation, the patient exhibited symptoms of severe hypokalemia and hypomagnesemia, attributed to medication non-adherence. Laboratory tests confirmed critically low potassium and magnesium levels, with elevated urine sodium and chloride. Treatment was initiated with oral and intravenous potassium and magnesium, leading to the normalization of electrolyte levels. This case highlights the challenges of managing GS, particularly in patients facing socioeconomic barriers that impede medication adherence and healthcare access. Personalized patient education, combined with comprehensive healthcare resources, is essential to mitigate complications and improve long-term outcomes in such cases. Full article

Several strategies, including UV detection with a diode array detector (DAD), laser-induced fluorescence (LIF), derivatization reactions, the use of micelles in the separation buffer, as well as online preconcentration techniques based on pressure-assisted electrokinetic injection (PAEKI), and offline preconcentration using solid-phase extraction (SPE) columns containing quaternary amine groups with a chloride counterion, were investigated for the simultaneous separation and signal amplification of free thyroid hormones (THs) in biological samples. Moreover, a sensitive method for the quantification of THs in selected biological samples using micellar electrokinetic capillary chromatography with LIF detection (MEKC-LIF) was developed. The THs present in biological samples (L-tyrosine, T2, T3, rT3, T4, and DIT) were successfully separated in less than 10 min. The analytes were separated following a derivatization procedure with fluorescein isothiocyanate isomer I (FITC). A background electrolyte (BGE) composed of 20 mM sodium tetraborate (Na₂B₄O₇) and 20 mM sodium dodecyl sulphate (SDS) was employed. Key validation parameters such as linearity, precision, limits of detection (LOD), and limits of quantification (LOQ) were determined. The use of PAEKI for the electrophoretic determination of free THs demonstrates significant potential for monitoring these hormones in real urine samples due to its high sensitivity and efficiency. Full article

Background: Different types of kidney stones are associated with distinct changes in urine chemistry. Methods: We assessed urinary parameters of 98 patients with calcium oxalate (CaOx) stones one month following endoscopic stone removal. The 24 h urine analysis encompassed the assessment of various parameters, including volume, sodium, chloride, sulfate, nitrate, fluoride, phosphate, calcium, potassium, magnesium, oxalate, uric acid, citrate, creatinine, and pH levels. Results: Hypocitraturia was the most prevalent urinary abnormality (61.2%, n = 63), followed by low urine volume (53%, n = 52) and hypercalciuria (50%, n = 49). We did not find any statistically significant differences between patients with whewellite (COM) (n = 69) and weddellite (COD) stones (n = 29) (p > 0.05). However, oxalate concentration was the only parameter with a statistically significant intergroup difference (p = 0.0297). Additionally, in univariate linear regression analysis, urinary phosphate levels ≥ 48.0 mmol/d showed a trend towards significance (OR 0.17, 95% CI 0.02–1.15, p = 0.0692), indicating that phosphaturia is associated with a significant increase in the odds ratio of COD stones. To further explore metabolic heterogeneity among stone formers, we conducted cluster analysis, which revealed three distinct metabolic subgroups. Cluster 1 was predominantly associated with COM stones (80.5%) and exhibited significantly higher urinary excretion of sodium, calcium, oxalate, phosphate, and uric acid compared to Cluster 2, which had a more balanced distribution of monohydrate and dihydrate stones. Conclusions: These findings suggest that a specific metabolic phenotype may be linked to COM stone formation, providing a framework for risk stratification and personalized prevention strategies in calcium oxalate stone formers. Full article

Background: Hyponatraemia is a rare but potentially life-threatening complication of terlipressin therapy. Case history: In the current case, a 39-year-old female with decompensated liver cirrhosis (Child-Pugh C) and acute variceal bleeding experienced a precipitous decline in serum sodium—from 136 mmol/L to 115 mmol/L—within 48 h of initiating terlipressin therapy. This was accompanied by marked fluid retention, reduced urine output, and symptoms of confusion and agitation. Laboratory tests confirmed dilutional hyponatraemia, characterized by urinary sodium <20 mmol/L and urine osmolality <100 mOsm/kg, indicating excessive free water reabsorption. Outcomes: The prompt discontinuation of terlipressin, fluid restriction and the cautious administration of hypertonic sodium chloride solution (2.7% NaCl) achieved a gradual normalization of sodium levels and resolution of symptoms. Fluid balance monitoring revealed a marked diuretic response following terlipressin cessation. This case aligns with existing reports, emphasizing the dual vasopressin receptor activity of terlipressin and its capacity to induce hyponatraemia, particularly in cirrhotic patients with preserved renal function and higher baseline sodium levels. Conclusions: This case and a literature review underscored the critical need for early fluid balance monitoring to detect retention. This case highlights the importance of individualized risk assessment, multidisciplinary management, and vigilant sodium correction to avoid complications. Practical recommendations are outlined to aid clinicians in the recognition and management of terlipressin-induced hyponatraemia. Full article

Background/Objectives: Temporal lobe epilepsy (TLE) often develops following an initial brain injury, where specific triggers lead to epileptogenesis—a process transforming a healthy brain into one prone to spontaneous, recurrent seizures. Although electroencephalography (EEG) remains the primary diagnostic tool for epilepsy, it cannot predict the risk of epilepsy after brain injury. This limitation highlights the need for biomarkers, particularly those measurable in peripheral samples, to assess epilepsy risk. This study investigated urinary metabolites in a rat model of TLE to identify biomarkers that track epileptogenesis progression across the acute, latent, and chronic phases and elucidate the underlying mechanisms. Methods: Status epilepticus (SE) was induced in rats using repeated intraperitoneal injections of lithium chloride–pilocarpine hydrochloride. Urine samples were collected 48 h, 1 week, and 6 weeks after SE induction. Nuclear magnetic resonance spectrometry was used for metabolomic analysis, and statistical evaluations were performed using MetaboAnalyst 6.0. Differences between epileptic and control groups were represented using the orthogonal partial least squares discriminant analysis (OPLS-DA) model. Volcano plot analysis identified key metabolic changes, applying a fold-change threshold of 1.5 and a p-value < 0.05. Results: The acute phase exhibited elevated levels of acetic acid, dihydrothymine, thymol, and trimethylamine, whereas glycolysis and tricarboxylic acid cycle metabolites, including pyruvic and citric acids, were reduced. Both the acute and latent phases showed decreased theobromine, taurine, and allantoin levels, with elevated 1-methylhistidine in the latent phase. The chronic phase exhibited reductions in pimelic acid, tiglylglycine, D-lactose, and xanthurenic acid levels. Conclusions: These findings highlight stage-specific urinary metabolic changes in TLE, suggesting distinct metabolites as biomarkers for epileptogenesis and offering insights into the mechanisms underlying SE progression. Full article

Metalloporphyrins (MPs) that induce heme oxygenase (HO)-1 were shown to attenuate complement-mediated glomerular injury, with cobalt protoporphyrin IX (CoPPIX) being the most effective. To decipher the efficacy between CoPPIX and its constituents (Co, PPIX), we compared the outcomes of treatment with each in a rat model of complement-dependent immune injury of glomerular epithelial cells (podocytes). Outcomes were correlated with HO-1 induction and expression levels of complement C3 and of the complement activation regulators (CARs) cluster of differentiation (CD)55, CD59, and CR1-related gene y protein product (Crry). Podocyte injury was induced in rats following a single injection of the complement-fixing antibody against the podocyte antigen, Fx1A. CoPPIX or its constituents, cobaltous chloride (CoCl₂) and protoporphyrin IX (PPIX), were injected prior to and on alternate days thereafter. Urine was assessed for protein excretion and kidney cortex samples were processed for histopathology and assessment of target gene mRNA and protein levels using digital polymerase Chain Reaction (dPCR) and capillary-based Western blot analysis. The anti-Fx1A antibody caused proteinuria and podocyte injury. Treatment with the full CoPPIX chelate reduced proteinuria but treatment with either CoCl₂ or PPIX did not. CoPPIX treatment potently induced HO-1 and reduced tissue C3 mRNA and protein levels. It also increased CD55, CD59, and Crry mRNA, with an inconsistent effect on protein levels. The Co moiety was required for HO-1 induction but not for the decrease in C3. This decrease did not significantly correlate with the effects of CoPPIX treatment on CD55 protein levels. Chelation of cobalt to PPIX enhanced its potency to induce HO-1 but reduced that on CD55 induction. These observations distinguish between the effects of CoPPIX and its constituents on proteinuria consequent to complement-mediated podocyte injury and underlying mediators and identify this MP as a potential disease-modifying agent. Full article

Background: Sodium, potassium, chloride, calcium, and magnesium in urine are useful biomarkers and are commonly evaluated in patients with different conditions. Urinary tract infections are among the most common diseases worldwide. However, their treatment poses significant challenges, particularly in hospitals, primarily due to antibiotic resistance and recurrence. Objectives: To evaluate the relationship between ion concentrations in urine and the presence of infection. Methods: A total of 175 random urine samples were collected from patients who had a request for urine culture at the Cova da Beira University Hospital Centre in Portugal. In vitro contamination was also conducted, in which ten negative urine cultures were contaminated with an Escherichia coli strain to evaluate the direct effect of its presence on the concentration of the ions. Results: In total, 61 samples were found to be positive, following a consensual quantitative definition. For Ca, there was a significant association between its concentrations in positive and negative cultures. In ten negative urine cultures experimentally contaminated with an Escherichia coli strain, bacterial growth did not seem to affect the concentration of ions. In vitro contaminated samples were also inoculated on MacConkey agar and incubated. The results showed that Gram-negative bacteria do not seem to proliferate in environments with low Ca concentrations. Conclusions: The presence of higher concentrations of Ca may facilitate the multiplication of Gram-negative bacteria, which can potentially result in depletion of Ca in vivo to putatively potentiate an inflammatory response. The concentration of Na, K, Cl, and Mg does not seem to have any relationship with UTIs. Full article

Background/Objectives: The antidiabetic effect of SGLT2 inhibitors (SGLT2-is) is based on their ability to increase glucose excretion through urine by inhibiting the kidney-resident SGLT2 protein. Euglycemic diabetic ketoacidosis (EuDKA) is an uncommon but potentially life-threatening adverse effect of these medications, which are notable for their antidiabetic, cardiovascular, and renal protective properties. This study aimed to clarify the impact of SGLT2-is on demographic, clinical, and biochemical characteristics in patients with DKA. Methods: A total of 51 individuals with a diagnosis of DKA were included in the trial; 19 of these patients were treated with SGLT2-is, while 32 were not. Patients diagnosed with DKA and treated with SGLT2-is were compared to those not treated with the medication in terms of clinical, biochemical, and laboratory characteristics. Results: The age of patients utilizing SGLT2-is was statistically considerably greater than that of non-users (p < 0.001). EuDKA was exclusively noted in the SGLT2-is cohort (p = 0.005). Urinary tract infections, vulvovaginitis, and genitourinary infections were substantially more prevalent among SGLT2-i users compared with non-users among both women and the overall patient group (p = 0.036, p = 0.001, p = 0.005, p = 0.003, respectively). Plasma glucose concentrations were significantly higher in SGLT2-i non-users (p = 0.006). Chloride (Cl⁻) concentrations were elevated among SGLT2-i users (p = 0.036). Conclusions: The study findings indicate that SGLT2 inhibitors may substantially influence age, serum chloride, EuDKA, and the occurrence of genitourinary infections in individuals with DKA. Full article

Urea, the end product of protein breakdown, is crucial for detecting chronic conditions. Normal urine urea levels range from 110 to 390 mM. This project aims to create a biosensor for rapid urea monitoring using urease immobilized on bovine serum albumin (BSA), safranin (SAF), and fullerene. The biosensor features a modified graphite paste electrode with a bionanocomposite, a silver chloride reference electrode, and a potentiostat set at +0.275 V. The sensor accurately measures urea concentrations from 68 to 410 mM in urine. Full article

The measurement of selected norepinephrine metabolites, such as 3,4-dihydroxyphenylglycol (DHPG), 3-methoxy-4-hydroxyphenylethylenglycol (MHPG), and vanillylmandelic acid (VMA), in biological matrices—including urine—is of great clinical importance for the diagnosis and monitoring of diseases. This fact has forced researchers to evaluate new analytical methodologies for their isolation and preconcentration from biological samples. In this study, the three most popular extraction techniques—liquid-liquid extraction (LLE), solid-phase extraction (SPE), and a new 3D-printed system for dispersive solid-phase extraction (3D-DSPE)—were investigated. Micellar electrokinetic chromatography (MEKC) with a diode array detector (DAD) at 200 nm wavelength was applied to the separation of analytes, allowing for the assessment of the extraction efficiency (R) and enrichment factor (EF) for the tested extraction types. The separation buffer (BGE) consisted of 5 mM sodium tetraborate decahydrate, 50 mM SDS, 15% (v/v) MeOH, 150 mM boric acid, and 1 mM of 1-hexyl-3-methylimidazolium chloride (the apparent pH of the BGE equaled 7.3). The EF for each extraction procedure was calculated with respect to standard mixtures of the analytes at the same concentration levels. The 3D-DSPE procedure, using DVB sorbent and acetone as the desorption solvent, proved to be the most effective approach for the simultaneous extraction and determination of the chosen compounds, achieving over 3-fold signal amplification for DHPG and MHPG and over 2-fold for VMA. Moreover, all extraction protocols used for the selected norepinephrine metabolites were estimated and discussed. It was also confirmed that the 3D-DSPE-MEKC approach could be considered an effective tool for sample pretreatment and separation of chosen endogenous analytes in urine samples. Full article

The sodium chloride cotransporter (NCC) is essential for electrolyte balance, blood pressure regulation, and pathophysiology of hypertension as it mediates the reabsorption of ultrafiltered sodium in the renal distal convoluted tubule. Given its pivotal role in the maintenance of extracellular fluid volume, the NCC is regulated by a complex network of cellular pathways, which eventually results in either its phosphorylation, enhancing sodium and chloride ion absorption from urines, or dephosphorylation and ubiquitination, which conversely decrease NCC activity. Several factors could influence NCC function, including genetic alterations, hormonal stimuli, and pharmacological treatments. The NCC’s central role is also highlighted by several abnormalities resulting from genetic mutations in its gene and consequently in its structure, leading to dysregulation of blood pressure control. In the last decade, among other improvements, the acquisition of knowledge on the NCC and other renal ion channels has been favored by studies on extracellular vesicles (EVs). Dietary sodium and potassium intake are also implicated in the tuning of NCC activity. In this narrative review, we present the main cornerstones and recent evidence related to NCC control, focusing on the context of blood pressure pathophysiology, and promising new therapeutical approaches. Full article

Background: In clinical practice, patient self-monitoring is crucial in achieving therapeutic goals in various diseases. In heart failure (HF), it is particularly important due to the increasing role of urine composition. Therefore, we proposed this study to assess the accuracy of urine chloride (uCl⁻) assessment via strip test in relation to chloride and sodium (uNa⁺) measurements in a gold-standard laboratory method. Methods: Urine samples were collected before administering morning medications. Afterwards, they were analyzed concurrently using the strip test and gold-standard laboratory method. Results: The study cohort comprised 66 patients (82% male, mean age 68 ± 12 years), of whom 65% were diagnosed with HF and 35% without HF. Across the entire cohort, a strong correlation was observed between uCl⁻ measured by both methods (r = 0.85; p < 0.001). However, the strip test was found to underestimate uCl⁻ relative to the laboratory measurements (mean difference of 18 mmol/L). Furthermore, strong correlations were observed between the methods among patients with HF and without HF (r = 0.88 vs. r = 0.71, respectively; p < 0.001 for both), where they presented similar relationship patterns. Interestingly, in patients with a low glomerular filtration rate (eGFR ≤ 60 mL/min/1.73 m²), the correlation between both methods was greater compared to those with high eGFR (>60 mL/min/1.73 m²) (r = 0.94 vs. r = 0.76, respectively; p < 0.001 for both). The relationship between uCl⁻ from the strip test and uNa⁺ from the laboratory measurement was weaker than for uCl⁻, but it was significant. Conclusions: These findings suggest that point-of-care strip tests for assessing urinary chloride demonstrate high accuracy and potential utility, particularly in patients with reduced eGFR. Full article

The objective of the present study was to compare principal cell-specific aquaporin-2 (AQP2) abundances in urinary extracellular vesicles (uEVs) on the first postoperative day in deceased-donor kidney transplant recipients without and with acute kidney injury. We measured uEV markers (CD9 and CD63) and the abundances of proximal tubular sodium-glucose transporter 2, distal tubular sodium/chloride cotransporter, and principal cell-specific aquaporin-2 using Western blotting of urine. uEV-AQP2 levels were normalized to living donor controls. The validation cohort consisted of 82 deceased-donor kidney transplant recipients who had a median age of 50 years (IQR 43 to 57 years). A total of 32% of recipients had acute kidney injury. The median uEV-AQP2 was significantly higher in recipients with acute kidney injury compared to immediate allograft function (2.05; IQR 0.87 to 2.83; vs. 0.81; IQR 0.44 to 1.78; p < 0.01). The Youden index indicated a uEV-AQP2 threshold of 2.00. Stratifying uEV-AQP2 into quartiles showed that recipients with higher uEV-AQP2 levels had higher rates of acute kidney injury (Cochran–Armitage, p = 0.001). The discovery cohort showed elevated CD9, CD63, and uEV-AQP2 levels in urine from recipients with acute kidney injury compared to immediate allograft function. We were able to quantify the damage of principal cells after kidney transplant to predict acute kidney injury using uEV-AQP2. Full article