Serum Sulfate Research Articles

Colorimetric detection of neomycin sulfate in serum based on ultra-small gold nanoparticles with peroxidase-like activity

Neomycin sulfate (NEO) is a kind of aminoglycoside antibiotics. Because of its strong ototoxicity, nephrotoxicity and other side effects, its content in the body should be strictly monitored during use. In this paper, a rapid colorimetric detection method for NEO based on ultrasmall polyvinylpyrrolidone modified gold nanoparticles (PVP/Au NPs) with peroxidase-like activity was developed. Firstly, ultra small PVP/Au NPs with weak peroxidase-like activity were synthetized. When they were mixed with NEO, strong hydrogen bonds were formed between NEO and PVP, resulting in the aggregation of PVP/Au NPs, and the aggregated PVP/Au NPs showed stronger peroxidase-like activity. Therefore, rapid colorimetric detection of NEO was achieved by utilizing the enhanced peroxidase-like activity mechanism caused by the aggregation of ultra small PVP/Au NPs. The naked eye detection limit of this method is 50 nM. Within the range of 1 nM-300 nM, there was a good linear relationship between NEO concentration and the change in absorbance intensity of PVP/Au NPs-H2O2-TMB solution at 652 nm, with the regression curve of y = 0.0045x + 0.0525 (R2 = 0.998), and the detection limit is 1 nM. In addition, this method was successfully applied to the detection of NEO in mouse serum. The recoveries were 104.4 % −107.6 % compared with HPLC assay results, indicating that this method for NEO detection based on PVP/Au NPs has great potential in actual detection of NEO in serum.

#2194 Targeted metabolomics analysis identifies a specific metabolomic profile in patients with early chronic kidney disease

Abstract Background and Aims The complex interconnections between various metabolites and the kidney play a major role in the pathogenesis of chronic kidney disease (CKD). Hence, by the assessment of the relationship between metabolites and the kidney, more accurate biomarkers could be discovered to prevent and detect CKD at an early stage. The primary goal of this study was to find new serum and urine biomarkers for the diagnosis of early CKD by conducting a metabolomic profiling through targeted metabolomics of both blood and urine in patients with CKD. Method This cross-sectional study comprised 80 patients with CKD who were categorized into six subgroups (G1-G6) based on their eGFR according to the KDIGO Guidelines. Additionally, 20 healthy individuals were included as healthy control participants (group C). Ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry was used to perform serum and urine metabolomic profiling. The 6 metabolites evaluated by this study were selected based on the results of a previous untargeted analysis research. Both blood and urine samples underwent multivariate analysis, which was subsequently followed by univariate analysis and quantitative analysis. Initially, the PLSDA score plot and VIP score were utilized. The application of the cross-validation procedure to the initial set of 6 molecules provided outstanding results, including high accuracy, high R² values, and a significant Q2 value. Hence, the model might be deemed as predictive. Random Forest analysis was utilized to conduct biomarker analysis and prediction. The specificity and sensitivity of the compounds identified as possible biomarkers were assessed using the Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC). Results There were strong positive correlations between eGFR and serum levels of Arginine (p < 0.05) and L-Acetylcarnitine (p < 0.05). Of note, the amounts of L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate in serum showed a progressive increase from group C to subgroups G1-G5. Furthermore, L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate exhibited a negative correlation with eGFR (p < 0.05). In addition, there was a positive correlation between the levels of urine Arginine, L-Methionine, and L-Phenylalanine and eGFR (p < 0.05). Urine levels of Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine were higher in groups G4 and G5, as compared to groups G1-G3b and C, respectively. The urinary concentrations of Indoxyl sulfate, Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine exhibited a negative correlation with eGFR (p < 0.05). Conclusion The current study shows that serum and urinary metabolites levels in patients with CKD display an alteration in production, secretion, and excretion in all CKD stages. There was demonstrated a distinct metabolomic profile in the course of CKD which points to novel biomarkers useful for the diagnosis and progression evaluation of CKD from its early stages.

Analytical methods for quantitating sulfate in plasma and serum.

Circulating sulfate needs to be maintained at sufficiently high levels for healthy growth and development. Animal studies have shown the adverse physiological consequences of low circulating sulfate level on the skeletal, neurological and reproductive systems. However, sulfate is not routinely measured in clinical investigations, despite the importance of sulfate being documented over the past several decades. Several methods have been developed for measuring serum and plasma sulfate level in animals and humans, including a range of barium sulfate precipitation techniques that have been a major focus of sulfate analytics since the 1960s. Evaluation of an ion chromatography method demonstrated its utility for investigation of sulfate levels in human health. More recently, liquid chromatography-tandem mass spectrometry has been used to show hyposulfatemia in a human case of mild skeletal dysplasia. This article provides an overview of analytical methods for measuring sulfate in serum and plasma, highlighting the strengths and limitations of each method.

Functional activity of the adrenal glands in type 2 diabetes patients with different hormonal and metabolic phenotypes

Background. Clinical trials showed a number of additional phenotypes of metabolic syndrome. All of them differ in the type of metabolic disorders and the composition of subcutaneous and visceral adipose tissue. Some of phenotypes have a number of clinical and metabolic similarities with endogenous or exogenous hypercorticism syndromes. The purpose was to characterize the functional activity of the adrenal glands in type 2 diabetes depending on the phenotypic features: the degree of general obesity and the level of visceral fat. Material and methods. Our trial included 89 patients with type 2 diabetes (46 men and 43 women) aged 32 to 85 years. The examination included evaluation of anthropometric parameters, body composition by the bioelectrical impedance method, assessment of the lipid and carbohydrate metabolism, the level of cortisol, dehydroepiandrosterone sulfate (DHEAS) in blood serum, and the activity of 11-beta-hydroxysteroid dehydrogenase (11β-HSD) enzyme. Results. Insulin and C-peptide levels were significantly lower in the non-obese group. The cortisol/DHEAS ratio was elevated in both subgroups with high levels of visceral fat as possible marker of imbalance of anabolic and catabolic hormones. In addition, the concentration of the cortisol, measured in the blood serum of the patients after waking up, was within the normal range. However, the average value in both groups was closer to its upper ranges. This may suggest the presence of subclinical hypercortisolism caused by an increased activity of 11β-HSD, which contributes to the local production of cortisol in visceral adipose tissue. Conclusions. The hormonal and metabolic changes that we found in our groups of patients with type 2 diabetes may indicate anabolic-catabolic imbalance, which is manifested both in the features of the topography of adipose tissue and in changes of metabolic processes, i.e. form the special metabolic phenotype with a catabolic or anabolic axis. Detection the subgroups at high risk allows to develop pathogenetic approaches to the most targeted comprehensive correction of existing violations.

A liquid chromatography-tandem mass spectrometry method for the analysis of dehydroepiandrosterone sulphate (DHEAs) in serum and plasma with comparison to an immunoassay method in a neonatal population

The measurement of dehydroepiandrosterone-sulphate (DHEAs) is an important second-line test to aid in the diagnosis of premature adrenarche, peripubertal gynaecomastia in males and in identifying the source of elevated androgens in females. Historically, DHEAs has been measured by immunoassay platforms which are prone to poor sensitivity and more importantly poor specificity. The aim was to develop an LC-MSMS method for the measurement of DHEAs in human plasma and serum, develop an in-house paediatric (<6 year old) reference limit and compare the performance against the Abbott Alinity DHEAs immunoassay method.Following pre-treatment with an internal standard, samples were loaded onto EVOLUTE® EXPRESS ABN plate. Analytes were separated with reverse-phase chromatography using ACQUITY® UPLC® HSS T3 2.1 mm × 50 mm, 1.8 μm column. Mass spectrometry detection was performed using a Waters® Xevo TQ-XS in electrospray negative mode. For the paediatric reference range, samples were collected from an inpatient setting (age ≤ 6 years old) with no evidence of adrenal dysfunction or history of/current steroid use. The method comparison was performed using samples from this cohort aged between 0 and 52 weeks.The assay demonstrated linearity up to 15 µmol/L (r2 > 0.99) with a functional sensitivity of 0.1 µmol/L. Accuracy results revealed a mean bias of 0.7% (−14% to 15%) when compared against the NEQAS EQA LC-MSMS consensus mean (n = 48). The paediatric reference limit was calculated as ≤ 2.3 µmol/L (95% C.I. 1.4 to 3.8 µmol/L) for ≤ 6 year olds (n = 38). Comparison of neonatal (<52 weeks) DHEAs with the Abbott Alinity revealed that the immunoassay ran at a 166% positive bias (n = 24) which appeared to lessen with increasing age.Described is a robust LC-MSMS method for the measurement of plasma or serum DHEAs validated against internationally recognised protocols. Comparison of paediatric samples of <52 weeks against an immunoassay platform demonstrated that in the immediate new-born period results generated from the LC-MSMS method offer superior specificity than an immunoassay platform.

The pathogenetic role of glycoproteins and proteoglycans in dog glomerulonephritis

The aim: to determine the main pathogenetic links of glomerulonephritis in dogs with the participation of connective tissue biopolymers.&#x0D; Materials and methods The research was conducted by analyzing the sources of scientific literature (PubMed, Elsevier, electronic resources of the V. I. Vernadskyi National Library) and the clinical experience of the authors, thanks to which a scheme of the pathogenesis of glomerulonephritis in dogs with the participation of connective tissue biopolymers was developed.&#x0D; Results. According to the results of our research, it was established, that chronic glomerulonephritis in dogs is accompanied by neutrophilia, lymphocytosis, as well as urinary and nephrotic syndromes, the progression of which causes a violation of the functional state of the kidneys and liver. Sick dogs develop a nephrotic symptom complex – persistent proteinuria, hypoalbuminemia, dysproteinemia, and hyperlipidemia. Clinically, edema was not observed, because it is known, that in dogs they are rarely detected with nephrotic syndrome. Inflammation in the kidneys is accompanied by an increase in the concentration of glycoproteins and sialic acids in the blood serum. After treatment, there was a decrease in the inflammatory process in the kidneys, which was manifested by a decrease in neutrophilia and lymphocytosis, as well as the content of glycoproteins and sialic acids in the blood serum. The content of total chondroitin sulfates and the fractional composition of glycosaminoglycans did not change, and the level of excretion of oxyproline and uronic acids decreased compared to the indicators before the start of treatment. This, in our opinion, is due to the slowing down of fibrotic processes in the kidneys. Thus, biochemical indicators of the state of connective tissue in the blood serum and urine of dogs with glomerulonephritis allowed us to evaluate the functional state of the kidneys for their inflammation (oxyproline and uronic acids), as well as to determine the violation of proteoglycan synthesis in nephrotic syndrome.&#x0D; Conclusions. For glomerulonephritis in dogs on the background of depression, decreased appetite, polydipsia, pain during palpation in the lumbar region, periodic vomiting, neutrophilic leukocytosis, lymphocytosis, nephrotic syndrome (hypoalbuminemia, increase in α2- and β-globulins, cholesterol, β-lipoproteins), growth activity of ALT, AST, and alkaline phosphatase, a decrease in Veltman's test, hyperazotemia, hyperphosphatemia, proteinuria, microhematuria, and cylindruria, there is an increase in the content of glycoproteins, sialic acids, and chondroitin sulfates, as well as heparan sulfate in the blood serum of dogs. The increase in the blood serum of patients with canine glomerulonephritis, as a marker of connective tissue – glycoproteins, sialic acids, chondroitinsulfates, and urinary excretion of oxyproline and uronic acids is due to inflammatory and fibrotic changes in the basal membranes of the kidney glomeruli

Serum sulfate level and Slc13a1 mRNA expression remain unaltered in a mouse model of moderate vitamin D deficiency.

Sulfate is essential for healthy foetal growth and neurodevelopment. The SLC13A1 sulfate transporter is primarily expressed in the kidney where it mediates sulfate reabsorption and maintains circulating sulfate levels. To meet foetal demands, maternal sulfate levels increase by twofold in pregnancy via upregulated SLC13A1 expression. Previous studies found hyposulfataemia and reduced renal Slc13a1 mRNA expression in rodent models with either severe vitamin D deficiency or perturbed vitamin D signalling. Here we investigated a mouse model of moderate vitamin D deficiency. However, serum sulfate level and renal Slc13a1 mRNA expression was not decreased by a moderate reduction in circulating vitamin D level. We confirmed that the mouse Slc13a1 5'-flanking region was upregulated by 1,25(OH)2D3 using luciferase assays in a cultured renal OK cell line. These results support the presence of a functional VDRE in the mouse Slc13a1 but suggests that moderate vitamin D deficiency does not impact on sulfate homeostasis. As sulfate biology is highly conserved between rodents and humans, we proposed that human SLC13A1 would be under similar transcriptional regulation by 1,25(OH)2D3. Using an online prediction tool we identified a putative VDRE in the SLC13A1 5'-flanking region but unlike the mouse Slc13a1 sequence, the human sequence did not confer a significant response to 1,25(OH)2D3 in vitro. Overall, this study suggests that moderate vitamin D deficiency may not alter sulfate homeostasis. This needs to be confirmed in humans, particularly during pregnancy when vitamin D and sulfate levels need to be maintained at high levels for healthy maternal and child outcomes.

Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays.

Steroid analysis in clinical laboratories is dominated by immunoassays (IAs) that have a high sample turnover but are inherently limited in trueness, precision, and sensitivity. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has proved to be a far more capable tool, delivering better sensitivity, specificity, and the possibility of parallel analysis of multiple steroids and metabolites, providing the endocrinologist with more reliable and comprehensive diagnostic information. An LC-MS/MS assay with gradient elution over less than eight minutes and a one-step sample preparation combining protein precipitation with phospholipid removal of off-line solid-phase extraction was developed and validated. It allowed the quantification of 11-deoxycorticosterone (11-DOC), 11-deoxycortisol (11-DF), 17-OH-progesterone (17P), 21-deoxycortisol (21-DF), androstenedione (ANDRO), aldosterone (ALDO), corticosterone (CC), cortisol (CL), cortisone (CN), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), estradiol (E2), progesterone (PROG), and testosterone (TES) in human serum. Interday imprecision was generally better than 15%, trueness was proven by recovery experiments with ISO 17034-certified reference materials, proficiency testing (UK NEQAS), and measuring serum reference standards. In-house comparison against IVD-CE-certified immunoassays (IA) for 17P, ANDRO, CL, DHEAS, E2, PROG, and TES was conducted by assessing leftover routine patient samples and purpose-built patient serum pools. None of the compared routine IAs were meeting the standards of the LC-MS/MS. Insufficient overall comparability was found for ANDRO and 17P (mean bias > +65%). Accuracy limitations at lower concentrations were present in IAs for PROG, E2, and TES.

OR27-1 11-Oxygenated C19 Steroids Are the Predominant Androgens Responsible for Hyperandrogenemia in Cushing's Disease

Abstract Background Symptoms of hyperandrogenism are common in patients with Cushing's disease (CD), but they cannot be sufficiently explained by measured concentrations of circulating androgens. In this study we analyzed the contribution of 11-oxygenated (11oxC19) androgens to hyperandrogenemia in female patients with CD as well as the influence of treatment with steroidogenesis inhibitors osilodrostat and metyrapone on 11oxC19 and classic androgens. Methods In this single-center study, we assessed saliva day profiles of 23 females with treatment naïve CD, 26 female controls, 5 females with CD treated with metyrapone and 5 treated with osilodrostat for cortisol, cortisone, androstenedione (A4), 11-hydroxyandrostenedione (11OHA4), testosterone (T), 11-ketotestosterone (11KT) by by liquid chromatography tandem mass spectrometry as well as morning baseline levels of gonadotropins and estradiol, sex hormone-binding globulin, cortisol anddehydroepiandrosterone sulfate (DHEAS) in serum and adrenocorticotropic hormone in plasma. Results Treatment naïve females with CD showed significantly elevated area under the curve (AUC) of 11OHA4 and 11KT throughout the day compared to controls (11OHA4 mean rank difference (mrd) 18.13, p = 0.0002; 11KT mrd 17.42; p = 0.0005) whereas A4, T and DHEAS were comparable to controls. Patients with more symptoms of hyperandrogenism displayed higher concentrations of 11oxC19 androgens and had significantly lower SHBG concentrations. Gonadotropin levels were normal in all patients with CD (LH 7.18 U/l (SD 14.28 U/l); FSH 7.68 U/l (SD 12.0 U/l)) and did not correlate with any other parameters. Treatment with osilodrostat and metyrapone efficaciously blocked 11oxC19 synthesis. In metyrapone but not in osilodrostat treatment a trend towards increased concentrations of T and significantly increased A4-concentrations were observed (A4 mrd 23.07, p = 0.0119). Conclusion Hyperandrogenemia in CD is predominantly caused by excess of 11oxC19 androgens. Due to lower compensatory increase of A4 and T, osilodrostat seems to be more suitable for treatment of females with CD and hyperandrogenism than metyrapone. Presentation: Tuesday, June 14, 2022 9:45 a.m. - 10:00 a.m.

Shedding of the Endothelial Glycocalyx Independent of Systemic Tryptase Release during Oncologic Oral Surgery: An Observational Study.

The endothelial glycocalyx and endothelial surface layer are crucial for several functions of the vasculature. Damage to the glycocalyx (“shedding”) occurs during diverse clinical conditions, including major surgery. Mast cell tryptase has been proposed as one possible “sheddase”. During oncologic oral surgery, glycocalyx shedding could be detrimental due to loss of vascular barrier function and consequent oedema in the musculocutaneous flap graft. Concentrations of the glycocalyx components heparan sulphate and syndecan-1, as well as of tryptase in blood serum before and after surgery, were measured in 16 patients undergoing oncologic oral surgery. Secondary measures were the concentrations of these substances on postoperative days 1 and 2. Heparan sulphate rose from 692 (median, interquartile range: 535–845) to 810 (638–963) ng/mL during surgery. Syndecan-1 increased from 35 (22–77) ng/mL to 138 (71–192) ng/mL. Tryptase remained virtually unchanged with 4.2 (3–5.6) before and 4.2 (2.5–5.5) ng/mL after surgery. Concentrations of heparan sulphate and syndecan-1 in serum increased during surgery, indicating glycocalyx shedding. Tryptase concentration remained equal, suggesting other sheddases than systemic tryptase release to be responsible for damage to the glycocalyx. Investigating strategies to protect the glycocalyx during oncologic oral surgery might hold potential to improve flap viability and patient outcome.

11-Oxygenated C19 steroids are the predominant androgens responsible for hyperandrogenemia in Cushing's disease.

BackgroundSymptoms of hyperandrogenism are common in patients with Cushing’s disease (CD), yet they are not sufficiently explained by androgen concentrations. In this study, we analyzed the contribution of 11-oxygenated C19 steroids (11oxC19) to hyperandrogenemia in female patients with CD.MethodsWe assessed saliva day profiles in females with CD pre (n = 23) and post (n = 13) successful transsphenoidal surgery, 26 female controls, 5 females with CD treated with metyrapone and 5 treated with osilodrostat for cortisol, cortisone, androstenedione (A4), 11-hydroxyandrostenedione (11OHA4), testosterone (TS), 11-ketotestosterone (11KT), as well as metabolites of classic and 11-oxygenated androgens in 24-h urine. In addition, morning baseline levels of gonadotropins and estradiol, sex hormone-binding globulin, cortisol and dehydroepiandrosterone sulfate (DHEAS) in serum and adrenocorticotrophic hormone in plasma in patients and controls were investigated.ResultsTreatment-naïve females with CD showed a significantly elevated area under the curve of 11OHA4 and 11KT in saliva throughout the day compared to controls (11OHA4 mean rank difference (mrd) 18.13, P = 0.0002; 11KT mrd 17.42; P = 0.0005), whereas A4, TS and DHEAS were comparable to controls. Gonadotropin concentrations were normal in all patients with CD. After transsphenoidal surgery, 11oxC19 and their metabolites dropped significantly in saliva (11OHA4 P < 0.0001; 11KT P = 0.0010) and urine (11-oxo-androsterone P = 0.0011; 11-hydroxy-androsterone P < 0.0001), treatment with osilodrostat and metyrapone efficaciously blocked 11oxC19 synthesis.ConclusionHyperandrogenemia in CD is predominantly caused by excess of 11oxC19 steroids.

Quantitative analysis of specific androgens in serum and urine samples from male, pre, and postmenopausal subjects using LC-MRM-MS

Androgens are endogenous hormones that play a crucial role in the paracrine and intracrine hormone system to perform and maintain vital physiological functions. Altered levels of androgens are implicated in many diseases such as sexual dysregulation, breast cancer, prostate cancer, and heart diseases etc. In this manuscript we describe a liquid chromatography-mass spectrometry (LC-MS) method using multiple reaction monitoring (MRM) for quantitatively measuring specific androgens such as dehydroepiandrosterone, testosterone, androsterone sulphate, androstenedione, and dihydrotestosterone in serum and urine samples. Serum acquired from nine different subjects (three pre-menopausal women, three postmenopausal women, and three healthy males) were used to evaluate the developed methods. In the sample preparation methods for serum either protein precipitation or liquid–liquid extraction (LLE) was used while the analysis of urinary androgens used LLE. The extracted androgens were quantitatively measured using LC-MRM-MS to which known amounts of stable isotope labeled standards were added. This manuscript also presents a LC-MRM-MS method mode for the analysis of oxime derivatized androgens potentially to enhance the sensitivity of the assay if required, from urine and venous-drawn serum samples.

Ropivacaine with intraspinal administration alleviates preeclampsia-induced kidney injury via glycocalyx /alpha 7 nicotinic acetylcholine receptor pathway

ABSTRACT Preeclampsia is characterized by hypertension and proteinuria, which is associated with kidney injury. Glycocalyx (GCX) degradation mediated endothelial injury can result in proteinuria and kidney damage. alpha 7 nicotinic acetylcholine receptor (α7nAChR) connects nervous and immune systems to respond to stress or injury. We aimed to explore the protective effect and mechanism of intraspinal analgesia on maternal kidney injury in preeclampsia. Endotoxin-induced preeclampsia rats treated with ropivacaine via intraspinal administration. Renal histopathological examination was performed, cell apoptosis in the kidney, the levels of Glycocalyx markers of Syndecan-1 and heparin sulfate (HS) in maternal serum, Syndecan-1 along with α7nAChR in the kidney were measured. Our results showed that kidney injury was obviously in preeclampsia rats with proteinuria, endothelial damage, higher apoptosis rate, increasing levels of Syndecan-1 and HS in serum, upregulated Syndecan-1 expression but downregulated α7nAChR expression in kidney. Preeclampsia rats treated with intraspinal injected ropivacaine attenuated preeclampsia-induced kidney injury as Syndecan-1 and HS were decreased in serum, Syndecan-1 expression was suppressed as well as α7nAChR was activated in the kidney. Our results suggested that Ropivacaine administered through the spinal canal may protect preeclampsia-induced renal injury by decreasing GCX and α7nAChR activation.

Rare SLC13A1 variants associate with intervertebral disc disorder highlighting role of sulfate in disc pathology

Back pain is a common and debilitating disorder with largely unknown underlying biology. Here we report a genome-wide association study of back pain using diagnoses assigned in clinical practice; dorsalgia (119,100 cases, 909,847 controls) and intervertebral disc disorder (IDD) (58,854 cases, 922,958 controls). We identify 41 variants at 33 loci. The most significant association (ORIDD = 0.92, P = 1.6 × 10−39; ORdorsalgia = 0.92, P = 7.2 × 10−15) is with a 3’UTR variant (rs1871452-T) in CHST3, encoding a sulfotransferase enzyme expressed in intervertebral discs. The largest effects on IDD are conferred by rare (MAF = 0.07 − 0.32%) loss-of-function (LoF) variants in SLC13A1, encoding a sodium-sulfate co-transporter (LoF burden OR = 1.44, P = 3.1 × 10−11); variants that also associate with reduced serum sulfate. Genes implicated by this study are involved in cartilage and bone biology, as well as neurological and inflammatory processes.

A Liquid Chromatography-Tandem Mass Spectrometry Method for the Analysis of Dehydroepiandrosterone Sulphate (Dheas) in Serum and Plasma with Comparison to an Immunoassay Method in a Neonatal Population

A Liquid Chromatography-Tandem Mass Spectrometry Method for the Analysis of Dehydroepiandrosterone Sulphate (Dheas) in Serum and Plasma with Comparison to an Immunoassay Method in a Neonatal Population

One step derivatization and dispersive liquid-liquid microextraction of hydroxychloroquine sulfate for its sensitive and accurate determination using GC–MS

In the present study, a novel analytical method for the determination of hydroxychloroquine sulfate in human serum and urine samples was established. One step derivatization and dispersive liquid-liquid microextraction (DLLME) was developed for quantitative determination of hydroxychloroquine sulfate in aqueous samples. Hydroxychloroquine sulfate was first hydrolyzed and converted to its benzoate derivative by adding benzoyl chloride in chloroform which also served as extraction solvent. Significant parameters such as type/volume of extraction and dispersive solvents, concentration/volume of sodium hydroxide, type/period of mixing and concentration of derivatizing agent were carefully optimized by one variable at a time approach. Under the optimum DLLME conditions, limit of detection (LOD), quantitation (LOQ) and dynamic range were calculated as 35.2, 117.2 and 96–1980 μg/kg (ppb), respectively. Recovery studies were conducted by spiked human serum and urine samples and the results were ranged between 93 and 107% with low standard deviations. Developed method can be easily used in hydroxychloroquine sulfate based SARS-CoV-2 and malaria treatment studies.

Accurate and sensitive determination of hydroxychloroquine sulfate used on COVID-19 patients in human urine, serum and saliva samples by GC-MS

A rapid, accurate, and sensitive analytical method, ultrasonication-assisted spraying based fine droplet formation–liquid phase microextraction–gas chromatography–mass spectrometry (UA-SFDF-LPME-GC-MS), was proposed for the determination of trace amounts of hydroxychloroquine sulfate in human serum, urine, and saliva samples. To determine the best extraction strategy, several liquid and solid phase extraction methods were investigated for their efficiencies in isolation and preconcentration of hydroxychloroquine sulfate from biological matrices. The UA-SFDF-LPME method was determined to be the best extraction method as it was operationally simple and provided accurate results. Variables such as the extraction solvent, spraying number, sodium hydroxide concentration and volume, sample volume, mixing method, and mixing period were optimized for the proposed method using the one-variable-at-a-time approach. In addition, Tukey's method based on a post hoc comparison test was employed to evaluate the significant difference between the parameters inspected. After the optimization studies, the limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.7 and 2.4 μg/kg, respectively. The sensitivity of the GC-MS system based on the LOD was enhanced approximately 440-fold when the UA-SFDF-LPME method was employed. Spiking experiments were also conducted for the human serum, urine, and saliva samples to determine the applicability and accuracy of the proposed method. Recoveries for the human serum, urine, and saliva samples were found to be in the ranges of 93.9%–101.7%, 95.2%–105.0%, and 93.1%–102.3%, respectively. These results were satisfactory and indicated that the hydroxychloroquine sulfate level in the above biological samples could be analyzed using the proposed method.

Serum Sulphate Levels in Hemodialysis Patients.

Objective Sulphur, similar to phosphorus, is easily attached to organic compounds. The inadequate elimination of sulphate may cause high sulphate concentrations in hemodialysis (HD) patients because sulphate is low in free form in plasma. Although we are well aware of the accumulation of phosphorus in chronic dialysis patients, we do not have an adequate knowledge database about the sulphur compounds. This study was designed to determine the level of sulphate in hemodialysis patients. Materials and Methods Ninety-four prevalent HD patients and 33 patients without renal failure were included in the study. The serum inorganic sulphate levels were measured by turbidimetric technique. Moreover, the serum level of urea, creatinine, albumin, calcium, phosphorus, and parathyroid hormone concentrations was simultaneously recorded. Results Mean levels of plasma sulphate were significantly higher (0.56 ± 0.17 mM vs 0.31 ± 0.13 mM, p < 0.001) in HD patients. Serum sulphate level correlated with patient's age, serum albumin, serum BUN and creatinine, and serum phosphorus level in HD patients. Serum sulphate levels were not associated with serum parathyroid hormone levels. Conclusion Serum sulphate levels were approximately twofold higher in HD patients than in the normal control group. Inorganic sulphate does not seem to accumulate in long-term dialysis patients, and mild increased serum levels of sulphate has no poor clinical outcome in these patients.

SUN-004 THE REGULATION OF THE GUT-KIDNEY AXIS IN VIVO BY TOTAL FLAVONE OF ABELMOSCHUS MANIHOT, A NATURAL EXTRACT, CONTRIBUTES TO THE AMELIORATION OF ADENINE-INDUCED RENAL FAILURE

Targeting gut microbiota dysbiosis and uremic toxins accumulation is important for the amelioration of renal dysfunction in chronic renal failure (CRF). Total flavone of abelmoschus manihot (TFA), a natural extract, has been applied extensively for treatment of the CRF patients in China. However, its therapeutic mechanisms remained elusive. Therefore, in this study, using an adenine-induced renal failure (RF) rat model, we examined the effects of TFA on the gut-kidney axis in vivo, compared with lubiprostone (LUB), then to clarify the potential mechanisms. Rats were randomly divided into 4 groups, the Sham-operated group, the Vehicle-given group, the TFA-treated group and the LUB-treated group. TFA, LUB and saline were daily administrated for 4 weeks after induction of RF by adenine gavage with unilateral ureteral obstruction (UUO). Renal injurious and dysfunctional indicators, tubulointerstitial fibrosis (TIF), fecal microbiota, gut-derived uremic toxins, colon histological characteristics and tight junction proteins expressions, as well as the key protein expression levels of TLR4/NF-κB/NLRP3 inflammasome pathways in the colon and the kidneys were examined, respectively. In the RF model rats, in addition to renal lesions and uremic toxins accumulation, the abundance of Lactobacillus in the gut was decreased, while that of C. perfringens in the gut was increased; There appeared the ulcers in the colon, accompanied with the low-expression of ZO-1 and claudin-1; Besides, TLR4/NF-κB/NLRP3 inflammasome pathways in the colon and the kidneys were activated, respectively. After the treatment of TFA or LUB, renal dysfunction and TIF in the RF model rats were significantly improved. Furthermore, the disordered Lactobacillus, Enterococcus, E. coli and C. perfringens in the gut, the damaged intestinal environment and the accumulative indoxyl sulfate and p-cresyl sulfate in serum were all ameliorated. More notably, TFA synchronously inhibited TLR4/NF-κB/NLRP3 inflammasome pathways activity in the colon and the kidneys, whereas, LUB only suppressed TLR4/NF-κB/NLRP3 inflammasome pathway activity in the colon. TFA, as a natural regulator in vivo, regulates the gut-kidney axis and ameliorates renal dysfunction in the RF model rats by modulating gut microbiota, reducing uremic toxins and inhibiting TLR4/NF-κB/NLRP3 inflammasome pathways activity in the colon and the kidneys, which is different from LUB. These findings may provide the first evidence for the gut-kidney axis, as the unusual target of TFA treatment in CRF.

“Doctor, I do not understand the results of the test, because I swear I am not drinking alcohol.” Truth or lie?

Nowadays, alcohol consumption has still been a public health concern that frequently has forensic consequences. The measurement of markers of alcohol consumption or alcohol abuse is thus important to assess alcohol consumption or to confirm alcohol abstinence during the weeks or the months before the sampling, which can be decisive and guide drastically a court decision. Direct alcohol markers, such as ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS) in serum, EtG and fatty acid ethyl esters (FAEE) in hair, or phosphatidylethanol (PEth) in blood, are available markers with high specificity and sensitivity [1] . In some particular situations, elevation of the concentration of the marker is not the consequence of alcohol consumption by the patient, but the results of alcohol or marker exposition before sampling [2] . Thus, this could conduct to erroneous interpretation even if the result is not an analytical false-positive case. Recently, we have been confronted to several situations involving presence of PEth in blood of patients denying any alcohol consumption for a couple of months, which led us to look for a possible pitfall. Dried blood spots (10.5 μL) were obtained from three volunteers (two females and one male) after cleaning their hands with soap and water or disinfectant solution containing ethanol (70% or 98%) using Hemaxis ® (DBS System SA) kit. The PEth homolog 16:0/18:1 concentration was determined by LC-MS/MS (API 5000 Sciex) according to validated and published method [3] . The first volunteer (woman) declared to be teetotaler. PEth blood concentrations after washing her hands with Sterilium ® gel [ethanol 85% (w/w)] and ethanol solution [ethanol 98% (v/v)] were 130 ng/mL and 350 ng/mL, respectively. The second volunteer (woman) declared to consume alcohol < 60 g/day. PEth blood concentration after washing her hands with soap and water was 82 ng/mL. The PEth blood concentration after washing her hands with ethanol solution [ethanol 98% (v/v)] decreased to 430 ng/mL. The third volunteer (man) declared to consume alcohol < 60 g/day. PEth blood concentration after washing his hands with soap and water was 140 ng/mL. After washing his hands with ethanol solution [ethanol 98% (v/v)], the PEth blood concentration was 240 ng/mL. An abnormal elevation of PEth concentration in blood was observed after using ethanolic solution for hand cleaning. Thus, ethyl alcohol disinfection have to be avoid before blood sampling for DBS. If not, a rapid formation of PEth could happen during blood sampling, involving a risk of erroneous interpretation concerning alcohol consumption.