Amino Acid Profiling of Follicular Fluid in Assisted Reproduction Reveals Important Roles of Several Amino Acids in Patients with Insulin Resistance.

The global prevalence of insulin resistance (IR) is increasing continuously, influencing metabolic parameters and fertility. The metabolic changes due to IR can alter the molecular composition of plasma and other body fluids. Follicular fluid (FF) is derived mainly from plasma, and it is a critical microenvironment for the developing oocytes. It contains various metabolites and amino acids, and the quality of the oocytes is linked at least partially to amino acid metabolism. Our goal was to quantitatively determine the amino acid (AA) profile of FF in IVF patients and to compare IR and non-insulin resistance (NIR) groups to investigate the AA changes in their FF. Using UHPLC-based methods, we quantified the main 20 amino acids from human FF samples in the IR and NIR groups. Several amino acids (aspartate, glycine, glutamate, and cysteine) differed significantly (p < 0.05 or less) between the two groups. The most significant alterations between the IR and NIR groups were related to the glutathione metabolic pathway involving glycine, serine, and threonine. Since insulin resistance alters the amino acid composition of the FF, the oocytes may undergo metabolism-induced changes resulting in poor oocyte quality and less fertility in the insulin resistance groups.

Plasma Levels and Renal Handling of Amino Acids Contribute to Determination of Risk of Mortality or Feed of Ventilation in Patients with COVID-19.

COVID-19 infection may lead to serious complications, e.g., need for mechanical ventilation or death in some cases. A retrospective analysis of patients referred to our COVID Emergency Department, indiscriminately, was performed. A routine lab analysis measured amino acids in plasma and urine of patients. Data of surviving and deceased patients and those requiring or not requiring mechanical ventilation were compared, and logistic regression analyses have been performed. Deceased patients were older, had higher blood glucose, potassium, AST, LDH, troponin, d-dimer, hsCRP, procalcitonin, interleukin-6 levels (p < 0.05 for all). They had lower plasma serine, glycine, threonine, tryptophan levels (p < 0.01), higher tyrosine and phenylalanine levels (p < 0.05), and higher fractional excretion of arginine, methionine, and proline (p < 0.05) than survivors. In a regression model, age, severity score of COVID-pneumonia, plasma levels of threonine and phenylalanine were predictors of in-hospital mortality. There was a difference in ventilated vs. non-ventilated patients in CT-scores, glucose, and renal function (p < 0.001). Using logistic regression, CT-score, troponin, plasma level, and fractional excretion of glycine were predictors of ventilation. Plasma levels and renal excretion of certain amino acids are associated with the outcome of COVID-19 infection beside other parameters such as the CT-score or age.

New psychoactive versus conventional stimulants - a ten-year review of casework in Hungary.

New psychoactive stimulants appeared in Hungary in 2010 as in several other European countries. We present our findings from cases where new psychoactive and conventional stimulants (we listed amphetamine, methamphetamine and MDMA as conventional drugs) have been detected in biological specimens between 2010 and 2019. MATERIALS: Biological samples (including urine, blood and body tissues), sent to the Department of Forensic Medicine, University of Pécs, South-West Hungary, in the period 2010-2019. METHOD: High performance liquid chromatography coupled with diode array detection (HPLC-DAD); supercritical fluid chromatography coupled to tandem mass spectrometry (SFC-MS/MS). RESULTS: During the nine-year period between 2010 and 2019, we found new stimulants in 973 (21.1%) cases, and conventional stimulants in 658 (14.2%) cases (out of 4604 analyses -100%- of samples sent to the laboratory for toxicology screening). 594 (12.9%) of all cases were post mortem analyses. The new drugs we've detected could be classified into three groups based on their chemical structure: cathinones (in 960 from our cases), substituted phenethylamines (8), and tryptamines (5). The most frequently identified new psychoactive stimulants were (in the order of decreasing frequency): pentedrone (262), mephedrone (188), N-ethylhexedrone (126), methylenedioxypyrovalerone (MDPV; 98), α-pyrrolidinopentiophenone (alpha-PVP; 93), 4-CMC (35). CONCLUSION: The new substances were detected in highest proportion in 2011; by 2018, the number of conventional drugs exceeded the new stimulants in our cases. According to the data of the Hungarian seizures, the decrease was predictable: from 2015, the seizures of traditional stimulants exceeded the seizures of new stimulants. In 2019 the new stimulants were dominated again among the detected substances in the samples.

Novel automated immune turbidimetric assay for routine urinary cystatin-C determinations.

AIM: There is no commercially available urinary cystatin-C (u-CYSC) test in the market. Therefore, we optimized and validated an automated immune turbidimetric test for u-CYSC measurements and investigated u-CYSC concentrations in acute and chronic diseases which might lead to renal tubular disorders. MATERIALS & METHODS: A particle-enhanced immune turbidimetric assay was adapted and validated on a Cobas 8000/c502 analyzer. Urine samples of different patient groups were also analyzed. RESULTS: Our method showed excellent analytical performance. U-CYSC/u-creatinine (u-CREAT) was higher in sepsis-related acute kidney injury group (p < 0.001) compared with controls and to patients with chronic hypertension and Type 2 diabetes. CONCLUSION: We validated a fast, sensitive, fully automated u-CYSC assay which is ideal for routine use and might be a potential complementary laboratory test to evaluate renal tubular function.

Predictive value of serum gelsolin and Gc globulin in sepsis - a pilot study.

BACKGROUND: Simultaneous determination of the two main actin scavenger proteins in sepsis has not been investigated until now. In our pilot study, we elucidated the predictive values of Gc globulin and gelsolin (GSN) in sepsis by comparing them to classic laboratory and clinical parameters. METHODS: A 5-day follow-up was performed, including 46 septic patients, 28 non-septic patients and 35 outpatients as controls. Serum Gc globulin and GSN levels were determined by automated immune turbidimetric assay on a Cobas 8000/c502 analyzer. Patients were retrospectively categorized according to the sepsis-3 definitions, and 14-day mortality was also investigated. RESULTS: First-day GSN also differentiated sepsis from non-sepsis (AUC: 0.88) similarly to C-reactive protein (AUC: 0.80) but was slightly inferior to procalcitonin (PCT) (AUC: 0.98) with a cutoff value of GSN at 22.29 mg/L (sensitivity: 83.3%; specificity: 86.2%). Only first-day SOFA scores (0.88) and GSN (0.71) distinguished septic survivors from non-survivors, whereas lactate (0.99), Gc globulin (0.76) and mean arterial pressure (MAP) (0.74) discriminated septic shock from sepsis. Logistic regression analyses revealed SOFA scores and GSN being significant factors regarding 14-day mortality. First-day GSN levels were higher (p<0.05) in septic survivors than in non-survivors. Gc globulin levels remained higher (p<0.01) in sepsis when compared with septic shock during the follow-up period. CONCLUSIONS: Both serum GSN and Gc globulin may have predictive values in sepsis. Considering the small sample size of our study, further measurements are needed to evaluate our results. Measurement of Gc globulin and GSN maybe useful in assessment of sepsis severity and in therapeutic decision-making.

Validation of an automated immune turbidimetric assay for serum gelsolin and its possible clinical utility in sepsis.

BACKGROUND: Studies showing the potential predictive value of the actin-binding protein gelsolin, in critically ill patients are scarce. Moreover, even up to now a rapid automated measurement of gelsolin has still remained a challenge. Therefore, we developed and validated an automated serum gelsolin immune turbidimetric assay for possible clinical use. METHODS: Validation of serum gelsolin assay was performed on a Cobas 8000/c502 analyzer (Roche) according to the second edition of Eurachem guidelines. Furthermore, we also studied the diagnostic value of serum gelsolin in sepsis when investigating sera of septic (n = 25), systemic inflammatory response syndrome (SIRS; n = 8) and control patients (n = 14). We compared our previously published Western blot data with those of the new turbidimetric assay. RESULTS: The sample volume was 7 µL and the assay time was 10 minutes. The detection limit was 0.72 mg/L, intra- and inter-assay imprecision remained in most cases less than 5% expressed as CV. Recovery was found to be 84.56%-93.52% and linearity study gave an appropriate correlation coefficient by linear regression analysis (r2  = .998). Septic patients exhibited lower (P = .015) first-day serum gelsolin levels than SIRS patients, which confirmed our previous Western blot results. The determined cut-off point for serum gelsolin was 14.05 mg/L (sensitivity: 75%; specificity: 60%) when investigating its diagnostic value in sepsis. CONCLUSION: Based on the results, our immune turbidimetric measurement offers a rapid and accurate quantitation of gelsolin in human serum samples. Serum gelsolin seems a promising additional diagnostic marker of sepsis which has to be further investigated.

Urinary orosomucoid: validation of an automated immune turbidimetric test and its possible clinical use.

INTRODUCTION: Besides routine serum markers of inflammatory diseases, the diagnostic potential of selected urinary proteins has not been fully exploited yet. Former studies revealed that urinary orosomucoid (u-ORM) might have complementary information in inflammatory disorders. Our aim was to develop and validate a fully automated method for u-ORM measurements and to evaluate its potential clinical impact on systemic inflammatory diseases. MATERIALS AND METHODS: A particle-enhanced immune turbidimetric assay was validated for a Cobas 8000/c502 analyzer to determine u-ORM levels. Spot urine samples from 72 healthy individuals, 28 patients with Crohn's disease and 30 septic patients were studied. RESULTS: Our assay time was 10 minutes and the detection limit of u-ORM was 0.02 mg/L. The intra- and inter-assay imprecision expressed as CV was less than 5%, and the recovery ranged between 95-103%. Within 10 to 60 years of age, a preliminary reference range for urinary orosomucoid/creatinine ratio (u-ORM/u-CREAT) was found to be 0.08 (0.01-0.24) mg/mmol [median (2.5-97.5 percentiles)]. Compared to controls, a five-fold increase of u-ORM/u-CREAT values in Crohn's disease and approximately a 240-fold increase in sepsis were observed. CONCLUSIONS: We set up a fast, sensitive and precise turbidimetric approach for automated u-ORM determination. Our highly sensitive assay is ideal for routine u-ORM measurements and might be a potential novel laboratory test in the management of systemic inflammatory processes.

Embutramide, a Component of Tanax(®) (T-61) as a New Drug of Abuse?

Tanax(®) (T-61) is a euthanasia solution commonly used in veterinary medicine in Europe. It consists of three active components: embutramide, mebezonium iodide, and tetracaine hydrochloride. Human consumption of Tanax(®) (T-61) is usually associated with suicide attempts. In our 15-year-long practice, embutramide was detected only three times but within a short period. First, it was found in the urine of a 42-year-old veterinarian, and the other two observations were made in a 16-year-old young man. Urine samples were analyzed using Shimadzu Prominence TOX.I.S.II. HPLC-DAD system with online SPE extraction system. Both of the two patients denied any intention to die. These cases show that this veterinary drug may also be considered as potential drugs of abuse.

Simultaneous determination of 4-substituted cathinones (4-MMC, 4-MEC and 4-FMC) in human urine by HPLC-DAD.

This study developed a selective and rapid high-performance liquid chromatography-diode array detection method for the confirmation of different cathinone derivates in human urine. Samples were prepared by solid-phase extraction (SPE) using procaine hydrochloride as the internal standard. The chromatographic separation was performed on a Kinetex PFP column using isocratic elution. The mobile phase was composed of a mixture of acetonitrile (33%, v/v) and 0.005M ammonium trifluoroacetate buffer (67%, v/v; pH 4.93 ± 0.03) with a flow rate of 0.350 mL/min. The diode array detection was performed at 262 nm. The method was linear over the concentration range of 25-2,400 ng/mL. Intra-day and inter-day precision values for cathinones were less than 1.26% (relative standard deviation). The limit of detection for any compounds extracted from human urine by the optimized SPE method was 40 ng/mL and the limit of quantification was 100 ng/mL in the urine. The recovery rate of SPE was between 71 and 82% with a lower relative standard deviation than 2.35%.

Thin-layer chromatography-densitometry and liquid chromatography analysis of alkaloids in leaves of Papaver somniferum under stress conditions.

The effect of stress conditions on the concentrations of secondary metabolites were examined during various developmental stages of Papaver somniferum plants. P. somniferum plants were grown in laboratory conditions (Budakalász). The experiment consisted of 22 treatments. Significantly different alkaloid contents can be observed under different stress conditions. In general, the alkaloid contents of plants are very low; therefore, a highly sensitive and reliable method has to be developed for analysis. The amount of alkaloids was measured by 2 separation and detection techniques. Accuracy of the thin-layer chromatography method for quantitative analysis is limited. Without purification of samples the background is too noisy. Column liquid chromatography is a sensitive and relatively inexpensive method that allows precise quantitative determination of the alkaloid content.