Gas chromatography-mass spectrometry determination of nicotine and cotinine in urine: A study of the effect of passive smoking.

RATIONALE: Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS). METHODS: Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 µL was performed using GC-MS, and selected ion monitoring (SIM) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT. RESULTS: The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT. CONCLUSIONS: A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC-MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of samples on a daily routine basis.

Metabolic Fingerprint in Childhood Acute Lymphoblastic Leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy. Despite high cure rates, several questions remain regarding predisposition, response to treatment, and prognosis of the disease. The role of intermediary metabolism in the individualized mechanistic pathways of the disease is unclear. We have hypothesized that children with any (sub)type of ALL have a distinct metabolomic fingerprint at diagnosis when compared: (i) to a control group; (ii) to children with a different (sub)type of ALL; (iii) to the end of the induction treatment. MATERIALS AND METHODS: In this prospective case-control study (NCT03035344), plasma and urinary metabolites were analyzed in 34 children with ALL before the beginning (D0) and at the end of the induction treatment (D33). Their metabolic fingerprint was defined by targeted analysis of 106 metabolites and compared to that of an equal number of matched controls. Multivariate and univariate statistical analyses were performed using SIMCAP and scripts under the R programming language. RESULTS: Metabolomic analysis showed distinct changes in patients with ALL compared to controls on both D0 and D33. The metabolomic fingerprint within the patient group differed significantly between common B-ALL and pre-B ALL and between D0 and D33, reflecting the effect of treatment. We have further identified the major components of this metabolic dysregulation, indicating shifts in fatty acid synthesis, transfer and oxidation, in amino acid and glycerophospholipid metabolism, and in the glutaminolysis/TCA cycle. CONCLUSIONS: The disease type and time point-specific metabolic alterations observed in pediatric ALL are of particular interest as they may offer potential for the discovery of new prognostic biomarkers and therapeutic targets.

Dried urine spot (DUS) applied for sampling prior to the accurate HILIC-MS/MS determination of 14 amino acids.

Urine amino acid analysis has proven valuable for an array of clinical or nutritional studies. However, transportation of liquid urine sample shows certain disadvantages, such as possible leakage, need for cold chain and thus higher costs for their transport. Utilization of dried urine spots (DUS) can offer an interesting alternative. In the present study, a method was developed for the determination of 14 amino acids in DUS including the testing of in-house collection device and drying of the sample before analysis. Normal filter paper was tested as the means for sample collection. Absorption and extraction experiments were performed on 3 different types of filter paper, with 3 different extraction solvents and two different solvent volumes. The solvents used were mixtures of common analytical solvents (methanol, water, acetonitrile) using total volumes of 1 mL and 1.5 mL. Finally, 1 mL of acetonitrile: methanol: water 40:40:20 (v/v/v) was chosen as the optimal system. Analysis was performed on a UHPLC-MS system, using stable isotope labeled internal standards. Method validation included the study of limits of detection (LOD) and quantification (LOQ), linearity ranges, precision, matrix effect, extraction recovery, precision, and stability for each analyte. The obtained results were satisfactory, thus enabling application of the proposed method as an alternative to the analysis of liquid urine. Further utilization of DUS can offer advantages by enabling patient centric sampling even in long distances far from the analytical laboratories.

Analysis of Migrant Cyclic PET Oligomers in Olive Oil and Food Simulants Using UHPLC-qTOF-MS.

Oligomers are a particular category of non-intentionally added substances (NIAS) that may be present in food contact materials (FCMs), such as polyethylene terephthalate (PET), and consequently migrate into foods. Here, an ultra-high-pressure liquid chromatography quadruple time-of-flight mass spectrometry (UHPLC-qTOF-MS) method was developed for the analysis of 1st series cyclic PET oligomers in virgin olive oil (VOO) following a QuEChERS clean-up protocol. Oligomer migration was evaluated with two different migration experiments using bottles from virgin and recycled PET: one with VOO samples stored in household conditions for a year and one using the food simulant D2 (95% v/v ethanol in water) at 60 °C for 10 days. Calibration curves were constructed with fortified VOO samples, with the LOQs ranging from 10 to 50 µg L-1 and the recoveries ranging from 86.6 to 113.0%. Results showed no migration of PET oligomers in VOO. However, in the simulated study, significant amounts of all oligomers were detected, with the migration of cyclic PET trimers from recycled bottles being the most abundant. Additional substances were tentatively identified as linear derivatives of PET oligomers. Again, open trimer structures in recycled bottles gave the most significant signals.

UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies.

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% - 117%; repeatability was estimated in the range of 1.5% - 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.

Syncope without prodromes is associated with excessive plasma release of adenosine at the time of syncope during head-up tilt table test.

BACKGROUND: In syncopal patients without underlying structural disease, we sought to investigate the association of Adenosine Plasma Levels (ADP) with the clinical presentation of neurally mediated syncope (NMS) and the outcomes of Head-Up Tilt Table Test (HUTT) and Adenosine test (ADT). METHODS: We studied 124 patients with different clinical types of NMS, i.e., Vasovagal (VVS, n=58), non-prodromes (NPS, n=18), or situational syncope (SS, n=48), using a standard protocol including HUTT and ADT. During HUTT, ADP was measured in the supine position, at table tilting and in syncope. RESULTS: Baseline ADP did not differ among groups. ADP at syncope were higher in NPS (n=5) compared to VVS (n=20): 0.23 vs. 0.12 µΜ, p=0.03, and SS (n=22): 0.04 µΜ, p=0.02. In NPS, ADP increased from supine to syncope (n=5): 0.15 vs. 0.23 µΜ, p=0.04. In VVS, ADP increased only from supine to tilt position: 0.11 vs. 0.14 µΜ, p=0.02. In SS, ADP did not change during HUTT. In positive vasodepressor HUTT, ADP increased from supine to tilt position (p=0.002) and at syncope (p=0.01). In SS, 20.0% exhibited cardioinhibitory HUTT vs. 6.8% in other forms of syncope (p=0.04). In SS, 22.9% manifested positive ADT vs 6.6% in other types of syncope (p=0.012). CONCLUSION: The subset of NPS patients with positive HUTT, show excessive ADP release at the time of syncope. This may explain the lack of prodromes in this form of syncope. Such observations contribute to the understanding of distinct profiles of clinical forms of syncope and may differentiate the management approach accordingly.

Development, Validation and Application of an Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) Method after QuEChERS Cleanup for Selected Dichloroanilines and Phthalates in Rice Samples.

Dichloroanilines and phthalic acid esters (phthalates) are food contaminants, stable in solution even at high temperatures, which exhibit considerable toxic effects, while acting as endocrine disruptors. In the present study, a quick and easy UHPLC-MS/MS method for simultaneously analyzing two dichloroanilines (3,4-DCA and 3,5-DCA) and six phthalates (DMP, DnBP, BBP, DnOP, DEHP, and mBP) in commercial rice samples was developed, validated, and applied. For the cleanup process, the methodology of quick, easy, cheap, effective, rugged, and safe (QuEChERS) was applied, whereas different dispersants (GCB, C18, and PSA) were tested. What was developed and presented had limits of detection ranging from 0.017 up to 0.12 mg/kg, recoveries (trueness) below 120%, and relative standard deviations (RSD; precision) <15% for all target analytes, whilst no significant matrix effects occurred for all analytes. It was determined that the rice samples analyzed using this developed technique did not contain any of the two dichloroaniline compounds (3,4-DCA and 3,5-DCA) nor two of the six phthalate (DMP and mBP) compounds analyzed, while the levels of other phthalates (DEHP, BBP, DnBP and DnOP) were within the legal limits. The current method ensures a fast and easy approach for the high-throughput quantification of the selected food contaminants in rice.

Evaluation of Cocaine Effect on Endogenous Metabolites of HepG2 Cells Using Targeted Metabolomics.

Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging.

Development and validation of a RPLC-MS/MS method for the quantification of ceramides in human serum.

Ceramides are key-role lipids involved in numerous central cellular processes. A plethora of studies have demonstrated that the levels of ceramides in blood circulation are related to different disease states, such as type 2 diabetes, cardiovascular diseases, ovarian cancer, multiple sclerosis and others. Herein, a RPLC-MS/MS method for the rapid quantification of ceramides Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1) in human blood serum was developed and validated. Different sample preparation strategies including SLE, LLE and QuECheRS were tested with the aim to attain effective, accurate and reproducible determination of ceramides in serum samples. Intra and inter-day accuracy were found to be between 80.0-111% and 87.8-106%, respectively, for all ceramides, while intra and inter-day precision were found to vary from 0.05% to 10.2% %RSD and 2.2% to 14.0% %RSD, respectively. The lower limits of quantification were 2.3 ng/mL for Cer(d18:1/16:0) and Cer(d18:1/18:0) and 1.4 ng/mL for Cer(d18:1/24:0) and Cer(d18:1/24:1). The method was evaluated in accordance to bioanalytical method guidelines and was used for the determination of serum ceramides of patients with coronary artery disease to evaluate its utility in clinical analyses.

Diminished Systemic Amino Acids Metabolome and Lipid Peroxidation in Ureteropelvic Junction Obstruction (UPJO) Infants Requiring Surgery.

Congenital anomalies of the urinary tract, and particularly of obstructive nephropathy such as ureteropelvic junction obstruction (UPJO) in infants, can later lead to chronic kidney disease and hypertension. Fundamental questions regarding underlying mechanisms remain unanswered. The aim of the present study was to quantitate the systemic amino acids metabolome in 21 UPJO infants requiring surgery (Group A) and 21 UPJO infants under conservative treatment (Group B). Nineteen healthy age-matched infants served as controls (Group C). Serum amino acids involved in several pathways and representative metabolites, including the L-arginine-derived nitric oxide (NO) metabolites nitrite and nitrate and the lipid peroxidation biomarker malondialdehyde (MDA) were measured by gas chromatography-mass spectrometry (GC-MS) methods using their stable-isotope labeled analogs as internal standards after derivatization to their methyl esters N-pentafluoropropionic amides (amino acids) and to their pentafluorobenzyl derivatives (nitrite, nitrate, MDA). The concentrations of the majority of the biomarkers were found to be lower in Group A compared to Group B. Statistical analysis revealed clear differentiation between the examined study groups. Univariate statistical analysis highlighted serum homoarginine (q = 0.006), asymmetric dimethylarginine (q = 0.05) and malondialdehyde (q = 0.022) as potential biomarkers for UPJO infants requiring surgery. Group A also differed from Group B with respect to the diameter of the preoperative anterior-posterior renal pelvis (AP) as well as regarding the number and extent of inverse correlations between AP and the serum concentrations of the biomarkers. In Group A, but not in Group B, the AP diameter strongly correlated with hydroxy-proline (r = -0.746, p = 0.0002) and MDA (r = -0.754, p = 0.002). Our results indicate a diminished amino acids metabolome in the serum of UPJO infants requiring surgery comparing to a conservative group.

Development and validation of a fast gas chromatography mass spectrometry method for the quantification of selected non-intentionally added substances and polystyrene/polyurethane oligomers in liquid food simulants.

A simple, fast, sensitive and reliable method was developed for the simultaneous determination of 13 food contact materials (FCM) regulated substances and non-intentionally added substances (NIAS) migrating into official food simulants. The method has been optimized to quantify the monomers styrene and α-methyl styrene, selected polystyrene oligomers (dimers, trimers) and polyester urethane-based oligomers (PU) cyclic oligomers, as well as cyclic NIAS originating from food packaging such as 2,6-Di-tert-butylbenzoquinone and 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione. The method employs liquid-liquid extraction of aqueous ethanol food simulants with dichloromethane, and analysis with gas chromatography coupled to mass spectrometry (GC-MS) with a total analysis time of less than 16 min, with limits of detections ranging from 0.32 ng mL-1 (1,1-diphenyl-ethylene) to 14.8 ng mL-1 for 7,9-di-tert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione and respective limits of quantification from 1.0 ng mL-1 to 41.7 ng mL-1, for the same analytes. Accuracy and precision results showed that the method is sufficiently accurate for all target analytes, with recoveries ranging between 80 and 110% and relative standard deviations (RSDs) smaller than 16% at the three selected concentration levels. The method has been successfully applied to seven FCM. Results indicated that significant amounts of polystyrene monomers, dimers and trimers are migrating into food simulants; this is also the case for polyester urethane-based oligomers (PU). Exposure assessment estimation was performed using EFSA's approach on the total sum of migrating oligomers. In certain cases, amounts of PS and PU oligomers found to be in some cases higher than the respective limits, for the sum of oligomers with a MW lower than 1000 Da.

Development of a UHPLC-MS/MS method for the determination of 84 pharmaceuticals and drugs of abuse in human liver.

Analysis of post-mortem liver for toxicological reasons is a considerable option when blood is unavailable. The development of analytical methods for tissue specimens is not as straightforward as for biological fluids as tissue presents challenges to the analytical chemist. The present study reports the development of a UHPLC-MS/MS method for the detection and quantification of 84 drugs and pharmaceuticals in human liver. The selected target drugs include pharmaceutical drugs and drugs of abuse. Sample preparation was studied using QuEChERS and different ratios of solvent volume and sample mass. Best results were attained by homogenizing 1 g of liver with acetonitrile K2CO3 buffer (pH = 10), QuEChERS salts MgSO4/ NaCl (1st purification step) and PSA/ 150 mg MgSO4 (2nd purification step). The extracted sample was analysed on UHPLC-MS/MS in multiple reaction monitoring mode (MRM) on a reversed-phase (Acquity BEH C18) column. Elution was accomplished by gradient program of mobile phase A: water, 0.1% formic acid and B: methanol, 0.1% formic acid that lasted 17 min. The method was specific, without interferences from the complex matrix. Sensitivity was satisfactory with limit of detection (LOD) ranging from 0.01 ng/g to 4.94 ng/g. Validation study was based on the guidelines of international bodies and included evaluation of recovery, carry-over, matrix effect, accuracy, stability, and precision of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of liver tissue obtained post-mortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.

Virgin olive oil metabolomics: A review.

Metabolomics involvement in the study of foods is steadily growing. Such a rise is a consequence of the increasing demand in the food sector to address challenges regarding the issues of food safety, quality, and authenticity in a more comprehensive way. Virgin olive oil (VOO) is a key product of the Mediterranean diet, with a globalized consumer interest as it may be associated with various nutritional and health benefits. Despite the strict legislation to protect this high added-value agricultural commodity and offer guarantees to consumers and honest producers, there are still analytical issues needing to be further addressed. Thus, this review aims to present the efforts made using targeted and untargeted metabolomics approaches, namely nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry-based techniques (mainly LC/GC-MS) combined with multivariate statistical analysis. Case-studies focusing on geographical/varietal classification and detection of adulteration are discussed with regards to the identification of possible markers. The advantages and limitations of each of the aforementioned techniques applied to VOO analysis are also highlighted.

Serum-Targeted HILIC-MS Metabolomics-Based Analysis in Infants with Ureteropelvic Junction Obstruction.

Ureteropelvic junction obstruction (UPJO) constitutes the predominant cause of obstructive nephropathy in both neonates and infants. Fundamental questions regarding UPJO's mechanism, assessment, and treatment still remain unanswered. The aim of the present study was to elucidate potential differences through serum metabolic profiling of surgical cases of infants with UPJO compared to both nonsurgical cases and healthy age-matched controls. Early diagnosis of renal dysfunction in this cohort based on highlighted biomarkers was the ultimate goal. Thus, serum samples were collected from 20 patients preoperatively, 19 patients with mild stenosis treated conservatively, and 17 healthy controls. All samples were subjected to targeted metabolomics analysis by hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC LC-MS/MS). Both univariate and multivariate statistical analyses were performed. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) score plots showed that the studied groups differed significantly, with a panel of metabolites, including creatinine, tryptophan, choline, and aspartate, distinguishing patients who required surgery from those followed by systematical monitoring as well as from healthy controls, showing high performance as indicators of UPJO disease.

Urine and fecal samples targeted metabolomics of carobs treated rats.

Ceratonia siliqua, known as the carob, is considered to be of high nutritional value and of great economic significance due to its unique composition. The beneficial effects of carob against cancer, metabolic syndrome, diabetes, diarrhea, hyperlipidemia and gastro esophageal reflux disease are only a few of its therapeutic actions. Metabolomics-based analysis provides an ultimate tool, for the deciphering of nutritional intervention derived metabolic alterations. In the present study, 16 male Wistar rats were treated with carob powder for a 15-day period. Fecal and urine samples were collected at 5 time points (0, 1, 5, 10 and 15 days). By the applied HILIC-MS/MS method, 63 and 67 hydrophilic metabolites were detected in the fecal and urine samples, respectively, including amino acids, organic acids, sugars, vitamins and other endogenous compounds. A clear group separation based on fecal metabolome was observed after 1 day and 15 days treatment, while only a mild differentiation at day 1 was observed based on urine metabolome. Twenty-one fecal metabolites were responsible for the separation including amino acids and their derivatives, vitamins and organic acids. However, only 7 metabolites were altered in rat urine samples. Metabolic alterations in fecal samples could be attributed to physiological and biochemical adaptations derived from the nutritional intervention. Fecal targeted metabolomics were proven to be suitable for uplifting and highlighting such alterations.

The Strong Antioxidant Sheep/Goat Whey Protein Protects Against mTOR Overactivation in Rats: A Mode of Action Mimicking Fasting.

Whey protein, a by-product of the cheese industry, can be putatively used as a functional food due to its beneficial health properties. The main objective of the present study was to assess in vivo the effect of a sheep/goat whey protein on the plasma amino acid profile and mammalian target of rapamycin (mTOR), a regulator of skeletal myogenesis. A control group was fed with a standard commercial diet while the experimental group received a standard commercial diet plus sheep/goat whey protein for 28 days. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to determine plasma amino acid levels while the expression of p70-S6 Kinase 1 (p70-S6K1) in liver and quadriceps muscles was quantified and used as a biomarker of mTOR activity. The results obtained showed a decrease in the levels of essential and branched-chain amino acids (BCAAs) in the experimental group. Furthermore, p70-S6K1 expression was decreased in the liver of rats consumed whey protein. In conclusion, the reduction of amino acid levels and the concomitant inactivation of mTOR imply that whey could potentially act protectively against disorders induced by mTOR overactivation. Intriguingly, this mode of action mimics fasting, an approach with established advantageous health effects.

Targeted LC-MS/MS for the evaluation of proteomics biomarkers in the blood of neonates with necrotizing enterocolitis and late-onset sepsis.

Late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) are severe life-threatening conditions for neonates. Accurate, early diagnosis and timely initiation of treatment are crucial. Non-specific overlapping clinical signs along with the non-sensitive/specific diagnostic tools set obstacles to speedy, trustful diagnosis including differential diagnosis. The objective of this study was to evaluate the potential of targeted LC-MS/MS proteomics in identifying diagnostic biomarkers of NEC or LOS. We conducted a prospective case-control study evaluating serum proteomics profiles of 25 NEC, 18 LOS, and an equal number of matched control neonates, over three sampling points. Eighty-three concatemers and synthetic peptides belonging to 47 protein markers of the two diseases were selected after thorough literature search. A novel selected reaction monitoring (SRM), LC-MS/MS method was developed for their analysis and evaluation as potential biomarkers. Multivariate and univariate statistical analyses highlighted significant proteins in differentiating LOS and NEC neonates and diseased from controls. Moreover, panels of proteins were tested for their ability to distinguish LOS from NEC and controls. We suggest two panels of three proteins each, exhibiting very high diagnostic value for LOS and excellent diagnostic performance at the critical LOS-NEC differentiation, reaching an AUC ROC value close to 1 (0.999). These panels constitute a valuable starting point for further validation with broader cohorts of neonates, aiming to improve the clinical practice. Graphical abstract ᅟ.

The Role of Sarcosine, Uracil, and Kynurenic Acid Metabolism in Urine for Diagnosis and Progression Monitoring of Prostate Cancer.

The aim of this pilot study is to evaluate sarcosine, uracil, and kynurenic acid in urine as potential biomarkers in prostate cancer detection and progression monitoring. Sarcosine, uracil, and kynurenic acid were measured in urine samples of 32 prostate cancer patients prior to radical prostatectomy, 101 patients with increased prostate-specific antigen prior to ultrasonographically-guided prostatic biopsy collected before and after prostatic massage, and 15 healthy volunteers (controls). The results were related to histopathologic data, Gleason score, and PSA (Prostate Specific Antigen). Metabolites were measured after analysis of urine samples with Ultra-High Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) instrumentation. Multivariate, nonparametric statistical tests including receiver operating characteristics analyses, one-way analysis of variance (Kruskal-Wallis test), parametric statistical analysis, and Pearson correlation, were performed to evaluate diagnostic performance. Decreased median sarcosine and kynurenic acid and increased uracil concentrations were observed for patients with prostate cancer compared to participants without malignancy. Results showed that there was no correlation between the concentration of the studied metabolites and the cancer grade (Gleason score <7 vs. ≥7) and the age of the patients. Evaluation of biomarkers by ROC (Receiving Operating Characteristics) curve analysis showed that differentiation of prostate cancer patients from participants without malignancy was not enhanced by sarcosine or uracil levels in urine. In contrast to total PSA values, kynurenic acid was found a promising biomarker for the detection of prostate cancer particularly in cases where collection of urine samples was performed after prostatic massage. Sarcosine and uracil in urine samples of patients with prostate cancer were not found as significant biomarkers for the diagnosis of prostate cancer. None of the three metabolites can be used reliably for monitoring the progress of the disease.

Targeted Metabolic Profiling of the Tg197 Mouse Model Reveals Itaconic Acid as a Marker of Rheumatoid Arthritis.

Rheumatoid arthritis is a progressive, highly debilitating disease where early diagnosis, enabling rapid clinical intervention, would provide obvious benefits to patients, healthcare systems, and society. Novel biomarkers that enable noninvasive early diagnosis of the onset and progression of the disease provide one route to achieving this goal. Here a metabolic profiling method has been applied to investigate disease development in the Tg197 arthritis mouse model. Hind limb extract profiling demonstrated clear differences in metabolic phenotypes between control (wild type) and Tg197 transgenic mice and highlighted raised concentrations of itaconic acid as a potential marker of the disease. These changes in itaconic acid concentrations were moderated or indeed reversed when the Tg197 mice were treated with the anti-hTNF biologic infliximab (10 mg/kg twice weekly for 6 weeks). Further in vitro studies on synovial fibroblasts obtained from healthy wild-type, arthritic Tg197, and infliximab-treated Tg197 transgenic mice confirmed the association of itaconic acid with rheumatoid arthritis and disease-moderating drug effects. Preliminary indications of the potential value of itaconic acid as a translational biomarker were obtained when studies on K4IM human fibroblasts treated with hTNF showed an increase in the concentrations of this metabolite.

The ovarian response to standard gonadotropin stimulation is influenced by AMHRII genotypes.

The aim of the current study was to explore whether anti-Müllerian hormone receptor II (AMHRII) genetic variants influence the hormonal profile and the ovarian response to standard gonadotropin stimulation of women undergoing medically assisted reproduction. Three hundred in vitro fertilization or intracytoplasmic sperm injection patients constituted the study population, while 300 women with at least one spontaneous pregnancy participated as controls. The follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and AMH levels were determined at the third day of the menstrual cycle. AMHRII 10A > G (rs11170555), 1749C > T (rs2071558) and -482A > G (rs2002555) polymorphisms were genotyped. The follicle and oocyte numbers, the follicle size and the clinical pregnancies were recorded. Regarding the AMHRII 1749C > T polymorphism, 1749CT women presented with higher total follicle and small follicle numbers compared to 1749CC women (p = 0.04 and p = 0.01, respectively). Whereas, as concerns the -482A > G polymorphism, -482AG women were characterized by higher total follicle and small follicle numbers comparing with -482AA women (p = 0.07 and p = 0.004, respectively). Finally, -482AG women presented with increased FSH levels compared to -482AA women (p < 0.05). However, no associations of AMHRII gene polymorphisms with serum AMH levels or clinical pregnancy rates were observed. AMHRII 1749C > T and -482A > G genetic variants were associated with the ovarian response to standard gonadotropin stimulation, affecting mainly the follicular growth.