Thin-layer Chromatography Assay Research Articles

The in vitro antioxidant properties of Bacillus simplex XJ-25 isolated from sand biological soil crusts

XJ-25, a strain with strong antioxidant activity was isolated from sand biological soil crusts in Gurban Tonggut Desert, Xinjiang, China. Strain XJ-25 is closely related to Bacillus simplex through the 16s rDNA sequencing combined with morphological, physiological and biochemical analysis. The medium for optimal antioxidant activity was NB with 1.5 g/L glucose. Based on the kinetic assay, antioxidant activity began at early exponential growth phases; maximum activity was reached at the stationary phase. Scavenging effects on 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), the hydroxyl and superoxide radicals, T-AOC capacity and protection against lipid damage were evaluated. The main antioxidant compounds were in the extracellular secreted supernatant of XJ-25. The active compounds were very stable at a pH range of 2 to 12, and temperature from 40 to 121°C, as well as in some organic solvents. Thin layer chromatography assay by DPPH scavenging assay showed two active spots with a Rf values of 0.35 and 0.47, and both of them were ninhydrin positive.

Propolis, a product of the bee hive, has an antagonistic affect on quorum‐sensing regulated bioluminescence, gene transcription and motility

Propolis or ‘bee hive glue’ is collected by bees from local flora and has been used to protect against virus, bacteria, inflammation and ulcers. The purpose of our study was to examine the effect of propolis on a bacterial gene regulatory process called quorum sensing (QS). QS regulatory activity of six samples of propolis was examined using five QS N‐acyl‐homoserine lactone (AHL)‐dependent bacterial biosensors. Concentrations of propolis that do not affect biosensor growth were exposed to the biosensors with inducing concentrations of the cognate AHL. Propolis antagonized the QS bacterial signaling system in liquid‐ and agar‐based bioassays and in C18 reverse‐phase thin‐layer chromatography assays. Using RT‐PCR, genes involved in QS signaling in pathogen Pectobacterium carotovorum were repressed by propolis. Pseudomonas aeruginosa, uses QS to conduct its swarming motility behavior. When propolis is added to P. aeruginosa, swarming motility is reduced up to 58.5%. Together, we present evidence that propolis samples contain compounds that suppress QS responses. In this regard, propolis could be the starting point for the discovery of novel anti‐pathogenic compounds to disrupt QS signaling systems.

Short communication: A sensitive method for qualitative screening of bile salt hydrolase-active lactobacilli based on thin-layer chromatography

A sensitive protocol based on thin-layer chromatography (TLC) was developed to screen qualitatively bile salt hydrolase (BSH)-active lactobacilli. The sodium salts of glycocholic acid and taurocholic acid were used as substrates, and bacterial BSH activity was confirmed by detecting cholic acid as a product of the bile conjugates using a TLC assay with direct visual observation. Forty-five lactobacilli isolated from human fecal samples were tested for BSH activity by the TLC assay, a conventional plate assay, and a quantitative colorimetric assay. With the TLC and quantitative colorimetric assays, the same 24 BSH-positive strains were detected. No false-positive or false-negative results were detected by the TLC assay. However, only 20 BSH-positive strains were detected with the conventional plate assay. Compared with the conventional plate assay, the TLC assay is more sensitive for the detection of BSH activity of lactobacilli and, thus, more suitable for screening of BSH-active lactobacilli of human origin.

Construction of an effective screening system for detection of Pseudomonas aeruginosa quorum sensing inhibitors and its application in bioautographic thin-layer chromatography

In Pseudomonas aeruginosa, quorum sensing (QS) regulates dozens of genes and proteins, many of which contribute to the virulence of this pathogen. QS inhibitory (QSI) compounds have been proposed as potential agents for treatment of bacterial infections. To search for Ps. aeruginosa QS inhibitors, we constructed an effective screening system, QSIS-lasI selector, based on the PlasI-sacB reporter, in which QS could be induced with 20nM 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide (3-oxo-C(12)-HSL). During screening of the crude extracts from 65 marine fungi, an isolate of Penicillium atramentosum was found to have QSI activity. Thin-layer chromatography assay of the fungal extracts for bioautographic identification of QSIS-lasI indicated that this fungus produced several QSI compounds, including QS inhibitors other than penicillic acid or patulin.

Bioassay-guided isolation and identification of antifungal components from propolis against Penicillium italicum

The present study was aimed at identification of antifungal components against Penicillium italicum from Chinese propolis with bioassay-guided fractionation technique. Propolis ethanolic extract (PEE) was separated and purified by liquid–liquid extraction and thin layer chromatography (TLC) and the most active band was subjected to HPLC–MS/MS to identify the antifungal compounds. The results showed PEE and its fractions had strong antifungal activity against P. italicum. Among the fractions of PEE partitioned by petroleum ether, ethyl acetate, n-butanol and water, ethyl acetate fraction (E-Fr) exhibited the most effective activity against P. italicum. Further bioautographic TLC assay showed Band I, with Rf value of 0.70, had an inhibitive zone, which showed the strongest antifungal activity and completely inhibited the growth of P.italicum at 200mg/L. Bioactive components found in Band I were further identified as pinobanksin, pinocembrine, chrysin and galangin. This study exhibited Chinese propolis and its main flavonoids was potential natural alternatives for the control of citrus blue mould caused by P.italicum.

Optimisation of a microwave‐assisted method for extracting withaferin A from Withania somnifera Dunal. using central composite design

Recently, there have been growing attention on the modification and optimisation of new extraction and quantification methods, caused by the lack of environmentally friendly methodologies for the extraction of phytochemicals from complex matrices. In the case of pharmaceutical compounds, not only the extraction procedure but also the analysis method should be efficient, precise, fast and easy. The essential pharmaceutical characteristics and trace concentration of withanolides led us to modify and optimise the previously reported extraction and quantification procedure for withaferin A (WA) as a candidate for withanolides. The WA from the air-dried aerial part of Withania somnifera Dunal. was extracted using a microwave-assisted extraction (MAE) technique. Four variables affecting the extraction procedure were optimised using the central composite design approach. The method of high-performance thin-layer chromatography assay was validated and applied for the quantification of each experiment. The optimum values of factors were: extraction time (150 s), extraction temperature (68°C) and 17 mL of methanol : water in the ratio 25 : 75 as extracting solvent. The solvent system consisted of ethyl acetate : toluene : formic acid : 2-propanol (7.0 : 2.0 : 0.5 : 0.5, v/v/v/v), and densitometric scanning at 220 nm was applied for the analysis. The dynamic linear range, LOD, LOQ and recovery with the inter-day, and intra-day RSDs of the developed method indicated the validity of the method. A pressurised MAE method for extracting WA from the plant's aerial part was optimised using factorial-based design. The net effect of time, temperature, solvent volume and its ratio suggests that the yield of WA increases until each factor reaches its optimum value, and decreases with further increase in temperature or solvent ratio.

English

Portulaca oleracea L. (POL) has been used as one of the traditional oriental medicines to treat bacteria, virus, antherasis, caducity, diabetes and for enhancing immunity. This study aims at revealing effects of POL on alloxan-induced diabetic rats and its mechanisms. According to thin layer chromatography assay, the main compounds of POL included organic acid,flavonoids, alkaloids, terpene ansteroid, hydroxybenzene, saponin and polysaccharide. The rats were divided into four groups: normal control (NC), diabetes control (DC), diabetes + POL high dose (400 mg/kg), diabetes + POLlow dose (200 mg/kg). The diabetic rats were administrated with POL or dH2Odaily for 28 days. The POL treatment resulted in significant decreases of fasting blood glucose, total cholesterol and triglycerides. POL also showed a tendency of improvement body weight gain on diabetic rats. Furthermore, the DC group had low serum insulin level comparing with that of NC group, at the same time,the insulin levels were dose-dependently raised in the POL-treated groups than that of DC group. According to single cell gel electrophoresis and LD50 analysis, POL was proved to be nontoxic to the animals. The results indicate that POL would alleviate the blood glucose and lipid rising associated with diabetes, and improve the abnormal glucose metabolism and increase insulin secretion byrestoring the impaired pancrease β cells in alloxan-induced diabetic rats, which suggest that POL has the hypoglycemic potential and could be useful on the diabetes therapy.   Key words: Portulaca oleracea L., diabetes, hypoglycemia, hypolipidemia, insulin.

Characterization of WbiQ: An α1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen

Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 ( DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an α1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-β1,3-GalNAc-α-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe, on a milligram scale.

Acetylcholinesterase Inhibitory Activity of Uleine from Himatanthus lancifolius

Application of acetylcholinesterase (AChE) inhibitors is the primary treatment for Alzheimer's disease. Alkaloids, such as physostigmine, galanthamine, and huperzine A, play an important role as AChE inhibitors. The aim of this work was to evaluate Himatanthus lancifolius (Muell. Arg.) Woodson, a Brazilian species of Apocynaceae, and its main indole alkaloid uleine, in order to identify new AChE inhibitors. The plant fluid extract, fractions, and uleine were tested for AChE inhibitory activity using Ellman's colorimetric method for thin-layer chromatography (TLC), 96-well microplates, and also Marston's TLC colorimetric method. Both TLC assays showed similar results. At 5 mg/mL, the fluid extract inhibited the AChE enzyme by (50.71 +/- 8.2)%. The ethyl acetate fraction exhibited the highest level of AChE inhibition, followed by the dichloromethane fraction. The isolated alkaloid uleine displayed an IC50 value of 0.45 microM.

Detoxification of aflatoxins by seed extracts of the medicinal plant, Trachyspermum ammi (L.) Sprague ex Turrill – Structural analysis and biological toxicity of degradation product of aflatoxin G1

Aqueous extracts obtained from leaves/seeds of various medicinal plants were evaluated for their ability to detoxify aflatoxin G1 (AFG1) by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Of the various plant extracts, the seed extract of Ajowan ( Trachyspermum ammi (L.) Sprague ex Turrill) showed the maximum degradation of AFG1 up to 65%. The dialyzed T. ammi extract was more effective than the crude extract, capable of degrading >90% of the toxin. The aflatoxin detoxifying activity of the T. ammi extract was drastically reduced upon boiling at 100 °C for 10 min. Significant levels of degradation of other aflatoxins viz., AFB1 (61%), AFB2 (54%) and AFG2 (46%) by the dialyzed T. ammi extract was also observed. Time course study of AFG1 detoxification by dialyzed T. ammi extract showed that more than 78% degradation occurred within 6 h and 91% degradation occurred 24 h after incubation. Mass spectral analysis of the degradation products of AFG1 revealed that the most abundant molecular ion peak in positive-ion mode was at m/z 288.29. MS/MS analysis of precursor ion at m/z 288.29 showed fragment transition to m/z 270.16 corresponding to the loss of 18 D. Mass spectral analysis of the degradation products of AFG1 suggests the modification of lactone ring structure. AFG1 caused more than 2% chromosomal aberration in corn at 40 mg/l concentration. However, AFG1 after treatment with T. ammi extract failed to induce chromosomal aberration demonstrating the degradation of AFG1 by T. ammi extract. The T. ammi extract may provide a biologically safe method to protect poultry or livestock feeds and other agricultural commodities from aflatoxins.

Therapeutic Drug Monitoring of Nevirapine in Resource‐Limited Settings

We developed a simple and inexpensive thin-layer chromatography (TLC) assay for semiquantitative detection of saliva concentrations of nevirapine in resource-limited settings. The method was validated in an African target population. Paired plasma and saliva nevirapine concentrations were assayed by high-performance liquid chromatography (HPLC); saliva concentrations of nevirapine were also assayed by TLC. The rate of false-positive results was the proportion of subtherapeutic nevirapine saliva and plasma concentrations determined by HPLC that were judged to be therapeutic in saliva specimens by TLC. The rate of false-negative results was the proportion of therapeutic nevirapine saliva and plasma concentrations determined by HPLC that were judged to be subtherapeutic in saliva specimens by TLC. The extent of agreement in TLC readings between 5 technicians and 2 batches of TLC sheets was evaluated. Twenty-five (9%) of 286 African adults had a subtherapeutic plasma nevirapine concentration. The median ratio of nevirapine concentrations in saliva to those in plasma was 0.51:1. The rate of false-positive results for TLC was 0% (0 of 23 specimens) when TLC results were compared with HPLC results for saliva specimens and 8% (2 of 25 specimens) when TLC results were compared with HPLC results for plasma specimens. The rate of false-negative results for TLC was 1% (3 of 263 specimens) when TLC results were compared with HPLC results for saliva specimens and 1% (3 of 261 specimens) when TLC results were compared with HPLC results for plasma specimens. The extent of agreement of TLC results was substantial for the 5 technicians (Fleiss's kappa = 0.77) and for the 2 batches of sheets (Cohen's kappa = 0.80). The TLC assay was found to be sensitive, specific, and robust in the detection of subtherapeutic nevirapine concentrations in saliva specimens obtained from African HIV-infected adults. It is an attractive alternative to HPLC for therapeutic drug monitoring of nevirapine in resource-limited settings.

A simple homogeneous scintillation proximity assay for acyl-coenzyme A:diacylglycerol acyltransferase

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol synthesis, and inhibiting this enzyme is a promising approach for treating obesity, type II diabetes, and dyslipidemia. There are two distinct DGAT enzymes: DGAT1 and DGAT2. The conventional assay for measuring DGAT activity is a thin layer chromatography (TLC) method, which is not amenable to screening a large number of compounds. To increase the throughput, we have developed a novel, homogeneous scintillation proximity assay (SPA) for DGAT. In this assay, when 3H-labeled acyl-CoA is used as the acyl donor and diacylglycerol is used as the acyl acceptor, the 3H-labeled triacylglycerol product formed in the reaction binds to polylysine SPA beads, producing a signal that is measured in a TopCount or LEADseeker. The apparent Michaelis–Menten kinetic parameters determined by this DGAT SPA method agreed well with the values determined with the conventional TLC assay. The statistical values also indicate that the DGAT SPA is a robust assay, with a Z′ of more than 0.60 and a signal/background ratio of approximately 9. These results suggest that the current assay provides high-throughput capacity for the identification of DGAT inhibitors.

The Human RecQ Helicases, BLM and RECQ1, Display Distinct DNA Substrate Specificities

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.

Hypoglycemic Effects and Mechanisms of Action of <i>Cortex Lycii Radicis</i> on Alloxan-Induced Diabetic Mice

Cortex Lycii Radicis (CLR) has been used as a traditional Oriental medicine as an antipyretic and to treat pneumonia, night-sweats, cough, hematemesis, inflammation, and diabetes mellitus for centuries. This study aimed to determine the effects of CLR on alloxan-induced diabetic mice and its mechanisms. Based on thin-layer chromatography (TLC) assay, the main compounds of CLR include an organic acid, flavone, alkaloid, polysaccharide, anthraquinone, and saponin. The mice were divided into four groups: normal control (NC), diabetes control (DC), diabetes+high-dose CLR (200 mg kg(-1)), and diabetes+low-dose CLR (100 mg kg(-1)). The diabetic mice were administered CLR daily for 28 days. The CLR treatment resulted in significant decreases in fasting blood glucose, total cholesterol, and triglycerides. CLR also showed a tendency to improve body weight gain in diabetic mice. Furthermore, the serum insulin level of each group was assayed, and the DC group had a lower serum insulin level than the NC group. Insulin levels were dose dependently raised in the CLR-treated groups compared with the DC group. According to single-cell gel electrophoresis and LD(50) analysis, CLR was nontoxic to the animals. The results indicate that CLR alleviates the blood glucose and lipid increases associated with diabetes and improves the abnormal glucose metabolism and increases insulin secretion by restoring impaired pancrease beta-cells in alloxan-induced diabetic mice. The results suggest that CLR has hypoglycemic potential and could be useful in diabetes therapy.

Diversity and quorum‐sensing signal production of Proteobacteria associated with marine sponges

Marine sponges are hosts to diverse and dense bacterial communities and thus provide a potential environment for quorum sensing. Quorum sensing, a key factor in cell-cell communication and bacterial colonization of higher animals, might be involved in the symbiotic interactions between bacteria and their sponge hosts. Given that marine Proteobacteria are known to produce N-acyl homoserine lactone (AHL) signal molecules, we tested the production of AHLs by Alpha- and Gammaproteobacteria isolated from marine sponges Mycale laxissima and Ircinia strobilina and the surrounding water column. We used three different AHL biodetection systems in diffusion assays: Chromobacterium violaceum, Agrobacterium tumefaciens and Sinorhizobium meliloti with optimal sensitivity to short-chain (C4-C6), moderate-chain (C8-C12) and long-chain (>or= C14) AHLs respectively. Thirteen of 23 isolates from M. laxissima and five of 25 isolates from I. strobilina were found to produce AHLs. Signals were detected from two of eight proteobacterial strains from the water column. Thin-layer chromatographic assays based on the A. tumefaciens reporter system were utilized to determine the AHL profiles of the positive isolates. The types and amounts of AHLs synthesized varied considerably among the strains. Small ribosomal rRNA gene sequencing revealed that the AHL-producing alphaproteobacterial isolates were mainly from the Silicibacter-Ruegeria subgroup of the Roseobacter clade. Two-dimensional gel electrophoresis (2DGE)-based proteomic analyses were congruent with phylogenetic relationships but provided higher resolution to differentiate these closely related AHL-producing strains.

Evaluation of Antioxidative Activity of Croatian Propolis Samples Using DPPH· and ABTS·+ Stable Free Radical Assays

Propolis is one of the richest sources of plant phenolics (flavonoids and phenolic acids), which are widely recognized as rather strong antioxidants. The aim of our work was to use colored stable free radical (DPPH* and ABTS*+) spectrophotometric and thin-layer chromatographic (TLC) assays to study the antioxidative behavior of the phenolics (caffeic acid, galangin and pinocembrin) most commonly present in Croatian propolis samples obtained from different Croatian regions. We propose a mathematical model providing a more sophisticated interpretation of the obtained results and a new parameter named antioxidative efficiency (AOE) is introduced. The kinetic behaviour of chosen standards determined by spectrophotometric assays follows the exponential decrease of the absorption curve. Explained numerically, AOE represents the absolute value of the first derivative of an absorbance curve in the point A0/e (where A0 is the absorbance measured at t = 0 and e is the natural logarithm base). The advantage of this newly introduced parameter is that it provides an easy and accurate mutual comparison between the rates of antioxidative efficiency of different propolis samples. A TLC assay was only applicable in the case of the DPPH* radical. Dose-response curves were described using a linear function with AOE expressed as a coefficient of the slope. The chromatographic method was shown to be very rapid, reliable and easy-to-perform.

Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules

2-Alkyl-4-quinolones (AHQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) are quorum sensing signal molecules. Here, we describe methods for AHQ detection, tentative identification and quantification, which employ a lux-based Pseudomonas aeruginosa AHQ biosensor strain. The protocol describes both thin-layer chromatography (TLC) and microtiter plate assays, which use bioluminescence or the green color of pyocyanin as detection end points. Organic solvent extracts of bacterial cells or cell-free culture supernatants are chromatographed on TLC plates, which are dried and overlaid with the AHQ biosensor. AHQs appear as both luminescent and green spots. For the microtiter assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AHQ biosensor. Light output is proportional to the AHQ content of the sample. The assays described take approximately 2 days to complete, are simple to perform, do not require sophisticated instrumentation and are highly amenable to screening large numbers of bacterial samples. However, apart from PQS and HHQ in P. aeruginosa, definitive AHQ identification will require additional MS and NMR analyses.

Development of a Simple and Robust Assay to Screen for Inhibitors of Sphingosine Kinases

Sphingosine kinases (SPHKs) catalyze the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), which plays important roles in a wide variety of intra- and extracellular functions. Conventionally, SPHK activity has been determined using radioisotope thin layer chromatography (TLC) and autoradiography to detect the product S1P. Here we describe the development of a simple and robust in vitro SPHK assay in 384-well format with no requirement for any separation steps such as extraction and TLC. The assay is based on (33)P-phosphate transfer from [gamma-(33)P]ATP to sphingosine and subsequent detection of the [(33)P]S1P using AquaBind plates (Asahi Techno Glass, Tokyo, Japan). Enzymatic and inhibition characteristics determined with this assay are in good agreement with previously reported values determined in the conventional TLC assay. K(m) values for D-erythro-sphingosine and ATP were determined to be 17.5 microM and 19.2 microM, respectively. The kinase reaction could be inhibited by ADP and N,N-dimethylsphingosine with a 50% inhibitory concentration of 410 microM and 450 microM, respectively. The established assay format was easily adapted to an automated screening platform and is characterized by a high signal-to-background ratio, small variation, and excellent Z factors.

Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans

Extracellular ATP and ADP trigger vasodilatatory and prothrombotic signalling events in the vasculature. Here, we tested the hypothesis that nucleotide turnover is activated in the bloodstream of exercising humans thus contributing to the enhanced platelet reactivity and haemostasis. Right atrial, arterial and venous blood samples were collected from endurance-trained athletes at rest, during submaximal and maximal cycle ergometer exercise, and after early recovery. ATP-specific bioluminescent assay, together with high-performance liquid chromatographic analysis, revealed that plasma ATP and ADP concentrations increased up to 2.5-fold during maximal exercise. Subsequent flow cytometric analysis showed that plasma from exercising subjects significantly up-regulated the surface expression of P-selectin in human platelets and these prothrombotic effects were diminished after scavenging plasma nucleotides with exogenous apyrase. Next, using thin layer chromatographic assays with [gamma-(32)P]ATP and (3)H/(14)C-labelled nucleotides, we showed that two soluble nucleotide-inactivating enzymes, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase, constitutively circulate in human bloodstream. Strikingly, serum nucleotide pyrophosphatase and hydrolase activities rose during maximal exercise by 20-25 and 80-100%, respectively, and then declined after 30 min recovery. Likewise, soluble nucleotidases were transiently up-regulated in the venous blood of sedentary subjects during exhaustive exercise. Human serum also contains 5'-nucleotidase, adenylate kinase and nucleoside diphosphate (NDP) kinase; however, these activities remain unchanged during exercise. In conclusion, intravascular ADP significantly augments platelet activity during strenuous exercise and these prothrombotic responses are counteracted by concurrent release of soluble nucleotide-inactivating enzymes. These findings provide a novel insight into the mechanisms underlying the enhanced risk of occlusive thrombus formation under exercising conditions.

Factitious Diarrhea Induced by Stimulant Laxatives: Accuracy of Diagnosis by a Clinical Reference Laboratory Using Thin Layer Chromatography

Surreptitious ingestion of laxatives can lead to serious factitious diseases that are difficult to diagnose. Most cases involve ingestion of bisacodyl or senna. Thin layer chromatography (TLC) of urine or stool is the only commercially available test for these laxatives. Such testing is considered highly reliable, but its accuracy in clinical practice is unknown. Our aim was to evaluate the reliability of TLC laxative testing by a clinical reference laboratory in the United States. Diarrhea was induced in healthy volunteers by ingestion of bisacodyl, senna, or a control laxative (n = 11 for each laxative group). Samples of urine and diarrheal stool were sent in blinded fashion to the clinical reference laboratory for bisacodyl and senna analysis. TLC testing for bisacodyl-induced diarrhea revealed a sensitivity of 73% and specificity of 91% when urine was tested and sensitivity and specificity of 91% and 96%, respectively, when stool was analyzed. When diarrhea was induced by senna, the TLC assay for senna failed to identify even a single urine or stool specimen as positive (zero% sensitivity). Considering the expected prevalence of surreptitious laxative abuse in patients with chronic idiopathic diarrhea (2.4%-25%, depending on the clinical setting), TLC of urine or stool for bisacodyl by this reference laboratory would often produce misleading results, and testing for senna would have no clinical value. The major problems are false-positive tests for bisacodyl and false-negative tests for senna.