Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Relationship between temperament and fatness in 11-year-old children and 17-year-old adolescents from Wrocław,Poland

Childhood obesity is increasing globally, and Poland is no exception. Studies indicate that relationship between obesity and psychological well-being is a complex issue and this needs further research. The objective of the present cross sectional study was to analyze the relationship between some temperament components and fatness among children in two developmental periods, approximately before and after adolescence. Participants included 122 children aged 11 years (57 boys and 65 girls), and 153 adolescents aged 17 years (64 boys and 89 girls) from 6 primary and 4 secondary schools in Wroc?aw, Poland. Height, weight, triceps-, subscapular and abdominal skinfold thicknesses were measured. Temperament was assessed by a questionnaire of Buss and Plomin (1984) in two versions: EAS-C for children completed by parents and EAS-D for youth and adults. Physical activity (PAL) was also assessed by a questionnaire. Mean, median and standard deviation (SD) were calculated and Student's t tests were performed to test for significance of differences between groups. Chi squared (χ²) statistic was used to test the significance of differences in frequencies. Analyses of covariance (ANCOVA) were performed to show the effect of the social - psychological factors on fatness. Controlling for socioeconomic status and physical activity level, activity, as a component of temperament had a significant effect on body fatness. The only component of temperament, which significantly influenced level of fatness in girls, was emotionality. These relationships differed according to sex and the two age groups concerned.     

Evaluation of the contribution of D9S1120 to anthropological studies in Native American populations

The D9S1120 locus exhibits a population-specific allele of 9 repeats (9RA) in all Native American and two Siberian populations currently studied, but it is absent in other worldwide populations. Although this feature has been used in anthropological genetic studies, its impact on the evaluation of the structure and genetic relations among Native American populations has been scarcely assessed. Consequently, the aim of this study was to evaluate the anthropological impact of D9S1120 when it was added to STR population datasets in Mexican Native American groups. We analyzed D9S1120 by PCR and capillary electrophoresis (CE) in 1117 unrelated individuals from 13 native groups from the north and west of Mexico. Additional worldwide populations previously studied with D9S1120 and/or 15 autosomal STRs (Identifier kit) were included for interpopulation analyses. We report statistical results of forensic importance for D9S1120. On average, the modal alleles were the Native American-specific allele 9RA (0.3254) and 16 (0.3362). Genetic distances between Native American and worldwide populations were estimated. When D9S1120 was included in the 15 STR population dataset, we observed improvements for admixture estimation in Mestizo populations and for representing congruent genetic relationships in dendrograms. Analysis of molecular variance (AMOVA) based on D9S1120 confirms that most of the genetic variability in the Mexican population is attributable to their Native American backgrounds, and allows the detection of significant intercontinental differentiation attributed to the exclusive presence of 9RA in America. Our findings demonstrate the contribution of D9S1120 to a better understanding of the genetic relationships and structure among Mexican Native groups.     

Kinetic characteristics and toxic effects of benzalkonium chloride following intravascular and oral administration in rats

Kinetic characteristics and toxic effects of benzalkonium chloride (BZK) following injection via jugular vein (JV), femoral artery (FA) and oral administration (PO) were experimentally investigated using rats. The BZK concentrations in blood and tissues (lung, liver and kidney) were determined by high-performance liquid chromatography with solid phase extraction. Toxic doses of 15 and 250 mg/kg of BZK were used for intravascular (JV and FA) and PO administration, respectively. The fatal effects appeared soon after the dose in JV-rats, while delayed in FA- or PO-rats. The blood BZK concentrations and the elimination half-lives were similar between JV- and FA-rats, while the distribution of BZK in tissues was slightly different. In PO administration, the rats that aspirated BZK into their lungs had some symptoms, while the rats that did not aspirate BZK appeared to be normal. The BZK concentrations in blood and tissues were significantly higher in the aspirated PO-rats. The toxic degree of BZK was correlated with the BZK concentration in orally dosed rats. Lung and kidney had higher BZK concentrations compared to blood or liver, and they could be the target organs of BZK.Keyword: Benzalkonium chloride     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Sex-specific reliability and multidimensional stability of responses to tests assessing neuromuscular function

The objective of this study was to estimate sex-specific effects in the test–retest cross-reliability of peripheral and central changes in nonlinear and linear measures of a surface electromyography signal during a brief (5 second) and sustained (2 minute) isometric maximal voluntary contraction, combined with superimposed electrical stimulation involving the ankle plantar flexors over five identical trials. In this study, we repeated the testing protocol used in our previous study of 10 women (age 20.9, SD = 0.3 years) (Bernecke et al., 2015) in a group of 10 men (age 21.2, SD = 0.4 years). Despite the central (sex effect; p < 0.05, η_p² > 0.71, SP > 70%) and peripheral fatigability (sex effect; p < 0.01, η_p² > 0.8, SP > 90%) during sustained isometric maximal voluntary contraction, and lower reliability for central activation ratio during brief (intraclass correlation coefficient [ICC] = 0.95 for men and ICC = 0.82 for women) and sustained maximal voluntary contraction (ICC > 0.82 for men and ICC > 0.66 for women) over ankle plantar flexors expressed in women more than in men, all the ICCs of all indices measured by tests assessing neuromuscular function across the five identical test–retest trials were found as meaningful (correlation significance of p < 0.05 was reached) and no significant differences were found between trials for any of the measured variables. In conclusion, the present study demonstrated greater central and peripheral fatigue for female participants following sustained (2 minute) isometric maximal voluntary contraction of the plantar flexor muscles for all repeated trials and indicated an acceptable agreement between measurements of the characteristic variables made using the three different devices (dynamometry, electrical stimulation, and surface electromyography) over time for both sexes.     

Antioxidant,anticancer, apoptosis properties and chemical composition of black truffle Terfezia claveryi

Desert truffles are seasonal and important edible fungi that grow wild in many countries around the world. Truffles are natural food sources that have significant compositions. In this work, the antioxidant, chemical composition, anticancer, and antiangiogenesis properties of the Terfezia claveryi truffle were investigated. Solvent extractions of the T. claveryi were evaluated for antioxidant activities using (DPPH, FRAP and ABTS methods). The extracts cytotoxicity on the cancer cell lines (HT29, MCF-7, PC3 and U-87 MG) was determined by MTT assay, while the anti-angiogenic efficacy was tested using ex-vivo assay. All extracts showed moderate anticancer activities against all cancer cells (p < 0.05). The hexane extract inhibited the brain cell line (U-87 MG) with an IC₅₀ of 50 μg/ml and significantly promoted cell apoptosis through the mitochondrial pathway and DNA fragmentation p < 0.001. The ethanol extract demonstrated potent antioxidants; DPPH, FRAP, and ABTS with an IC₅₀ value of 52, 48.5 and 64.7 μg/ml, respectively. In addition, the hexane and ethyl acetate extract significantly (p < 0.001) inhibited the sprouting of microvessels by 100% and 81.2%, at 100 μg/ml, respectively. The GC analysis of the most active extract (hexane) showed the presence of several potent phytochemicals such as stigmasterol, beta-Sitosterol, squalene, lupeol, octadecadienoic acid, and oleic acid.     

Corrigendum to “Dihydropyrimidine dehydrogenase: a flavoprotein with four iron–sulfur clusters” [Biochim. Biophys. Acta 1701 (2004) 61–74]

Lixelle is a direct hemoperfusion-type adsorption column that was developed to selectively eliminate β2-microglobulin (β2-m) from the circulating blood of patients with dialysis-related amyloidosis (DRA). The adsorbent in Lixelle comprises porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound. One milliliter of wet Lixelle beads eliminates more than 1 mg of β2-m in vitro. In hemodialysis patients who were treated with Lixelle, Lixelle improved joint pain, nocturnal awakening, pinch strength, motor terminal latency, and their activity of daily living. The adsorbent adsorbs β2-m selectively but not specifically, as well as inflammatory cytokines such as interleukin-1β and IL-6 which are considered to be involved in the development of DRA. Lixelle treatments reduce the circulating levels of β2-m and inflammatory cytokines, thereby improving the symptoms of patients with DRA.     

Analysis of N,N-diethyl-m-toluamide in porcine skin perfusates using solid-phase extraction disks and reversed-phase high-performance liquid chromatography

N,N-Diethyl-m-toluamide (DEET) is frequently used as an insect repellent by military and civilian populations. Because dermal exposure has resulted in several cases of DEET toxicosis, there is a need to rapidly and reliably determine DEET concentrations in biological matrices. An improved method for the analysis of DEET was developed for determining transdermal diffusion of low levels of DEET following application to an in vitro porcine skin flow-through diffusion cell system. The technical improvement involved the use of disk solid-phase extraction (SPE) instead of packed-bed SPE. The disk SPE method required small volumes of preconditioning, wash, and elution solvent (0.5-1 ml) to extract DEET from perfusate samples containing bovine serum albumin (BSA). The limit of quantitation (LOQ) was estimated as 0.08 micro g/ml DEET and recoveries from BSA media samples spiked with DEET ranged from 90.1 to 117% with relative standard deviation (RSD) ranging from 2.0 to 13.1%. This method was used to analyze perfusate samples from skin (n=4) topically exposed to DEET-ethanol formulations. The data from these analyses determined that DEET permeability in porcine skin was 2.55 x 10(-5)+/-0.54 x 10(-5) cm/h.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

Detection and determination of reticuline and N-methylcoculaurine in the Annonaceae family using liquid chromatography-tandem mass spectrometry

In Guadeloupe, the French West Indies, there is a high incidence of atypical parkinsonism or progressive supranuclear palsy, and all of the investigated patients had taken herbal tea or tropical fruits of the Annonaceae family. Local inhabitants consume the fruits, and also drink tea made from the leaves. In the present study, we used liquid chromatography-tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring (MRM) to detect low-molecular-weight neurotoxic benzylisoquinoline derivatives in the Annonaceae family. We detected reticuline and N-methylcoculaurine in every Annona muricata sample examined, except for pulp and seed. They were not detected in sweetsop fruits. Norreticuline was not detected in any sample. These three compounds were toxic to SH-SY5Y neuroblastoma cells and inhibited mitochondrial respiratory complex I. It is possible that uptake of the benzylisoquinoline derivatives reticuline and N-methylcoculaurine and their accumulation in the brain may be related to the pathogenesis of the local endemic disease.