Exploration of Raman exfoliated cytology for oral and cervical cancers

Exfoliated cell are one of the most patient compliant specimens, hence are workhorse of successful screening programs in cancers like cervix. Raman exfoliative cytology (REC), even on cell pellets using Raman microprobe, has demonstrated ∼80% specificity, comparable to conventional screening programs. Objective of this study is to further evaluate Raman spectroscopy, which is sensitive to biochemical composition, vis-a-vis conventional morphology based cytology that differentiates cell-type but not the origin. Exploration of this kind further corroborates utility and efficacy of REC as screening tool, also can pave way to prospective multi-cancer screening by a multi-group model. Therefore, the present study explores REC in a stringent two-cancer, four-group model for classification of 44 oral [29, tumor (OT), 15 normal (ON)] and 66 cervical (38 tumor (CT), 28 normal (CN)] exfoliated cell specimens. Spectra were acquired from cell pellets using Raman microprobe. Spectra were analyzed in two approaches, spectra-wise and subject-wise to circumvent inherent heterogeneity due to co-localization of normal cells in malignant specimens. In the first approach, PCA and LDA yielded a classification efficiency of 82% for OT and 77% for CT. A slightly higher classification (OT, 86% and CT, 84%) was achieved for the subject-wise approach. Thus findings of this study demonstrate similar efficacy and specificity to that of individual cancer-based models reported earlier. These findings are quite encouraging especially for resource deprived and under privileged countries where these two cancers are more prevalent and could be useful for prospective centralized REC facility.     

Proteomic identification of salivary transferrin as a biomarker for early detection of oral cancer

Oral cancer has a low five-year survival rate. Early detection of oral cancer could reduce the mortality and morbidity associated with this disease. Saliva, which can be sampled non-invasively and is less complex than blood, is a good potential source of oral cancer biomarkers. Proteomic analysis of saliva from oral cancer patients and control subjects was performed to identify salivary biomarkers of early stage oral cancer in humans. The protein profile of pooled salivary samples from patients with oral squamous cell carcinoma (OSCC) or OSCC-free control subjects was analyzed using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Potential biomarkers were verified by Western blotting and ELISA assays. Transferrin levels were elevated in the saliva of OSCC patients as determined using 2DE followed by MALDI-TOF MS and confirmed by MALDI-TOF/TOF MS, Western blotting and ELISA. The increase in salivary transferrin levels in OSCC patients strongly correlated with the size and stage of the tumor. The area under the receiver-operating characteristics curves showed that salivary transferrin-based ELISA was highly specific, sensitive and accurate for the early detection of oral cancer. We have identified salivary transferrin as a biomarker for the detection of early stage oral cancer. This finding provides a promising basis for the development of a non-invasive diagnostic test for early stage oral cancer.     

Screening for Oral Cancer Using Electrochemical Telomerase Assay

Electrochemical telomerase assay (ECTA) developed by our group was evaluated in an oral cancer screening using exfoliated oral cells and tissues obtained from patients of oral cancer, mucosa associated disease, or healthy volunteers. Telomerase activity from ECTA is correlated with hTERT mRNA expression level using a real‐time PCR and was increasing in the following order: healthy volunteer group<mucosa associated disease group<oral cancer group. Sensitivity and specificity of ECTA were 88 % and 72 %, respectively when used 17 % of the threshold value based on the receiver operating characteristic curve in ECTA data.     

Cancer,pre-cancer and normal oral cells distinguished by dielectrophoresis

Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C _Eff) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C _Eff of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C _Eff and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.     

Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions

A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15% of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12% of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.     

Validation of a screening method for corticosteroids in doping analysis by liquid chromatography/tandem mass spectrometry

A selective and sensitive method for the screening of nine corticosteroids in human urine has been validated. Analyses were performed using an ion trap instrument equipped with an electrospray ionisation (ESI) interface. All corticosteroids were separated in less than 20 min after liquid/liquid extraction with diethyl ether. The limit of detection for all substances was 4 ng/mL or lower. The method was applied to detect betamethasone after the intramuscular injection of Diprophos. Betamethasone could be detected for up to 12 days after administration. Validation of the chromatographic separation and mass spectrometric identification of mixtures of betamethasone and dexamethasone are also presented.     

Determination of geniposide in rat urine after oral administration of the traditional Chinese medicinal preparation yin-zhi-ku decoction by high-performance liquid chromatography

A high-performance liquid chromatography method with solid-phase extraction is introduced for the determination of geniposide in rat urine after oral administration of yin-zhi-ku decoction. Geniposide and an internal standard (paeoniflorin) are extracted from urine using Strata cartridges. Analysis of the extract is then performed on a reversed-phase C18 column using acetonitrile-water (14:86, v/v) as eluting solvent system. UV detection is set at 238 nm. The calibration curve for geniposide is linear (r = 0.9996) in the concentration range of 2.0-240 microg/mL. Both intra- and interday precision of the geniposide are determined, and their relative standard deviation does not exceed 10%. The validated method is successfully applied to determine geniposide from rat urine after oral administration of yin-zhi-ku decoction.     

The analysis of café espresso using two-dimensional reversed phase-reversed phase high performance liquid chromatography with UV-absorbance and chemiluminescence detection

In this study, an activity based screening technique combining two-dimensional liquid chromatography (2DHPLC) with UV-absorbance and chemiluminescence detection was applied to study “Ristretto”, “Decaffeinatto” and “Volluto” espresso coffees. This technique, which coupled the separation power of 2DHPLC with the sensitivity and selectivity of the chemiluminescence detection, offers great potential for screening complex samples for antioxidant compounds. Detailed information regarding the complexity of the sample, and the variation between these three coffees could be obtained using this multidimensional-hyphenated method of analysis.     

Screening of drugs of abuse and toxic compounds in human whole blood using online solid‐phase extraction and high‐performance liquid chromatography with time‐of‐flight mass spectrometry

A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid‐phase extraction with liquid chromatography coupled to time‐of‐flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid‐phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time‐of‐flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full‐scan (m/z 50–800) mode using an MS^E acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5–45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

High‐performance liquid chromatography with photodiode array detection and chemometrics method for the analysis of multiple components in the traditional Chinese medicine Shuanghuanglian oral liquid

Shuanghuanlian oral liquid, a traditional Chinese medicine preparation, is a mixture of three herbs (Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae). In this study, the quantitative analysis of three main active compounds, chlorogenic acid, forsythin and baicalin in samples from different manufacturers was performed rapidly by high‐performance liquid chromatography coupled with photodiode array detection followed by Contour Projection coupled to stepwise regression treatment of the obtained three‐dimensional spectra in which the partial overlap between adjacent target components existed. The method was validated for linearity (R>0.9940), precision (RSD<1.25%), recovery (92.20–102.50%), limit of detection (0.01–0.02 μg/mL) and limit of quantification (0.03–0.07 μg/mL). The results indicated that the combination of the three‐dimensional spectra of traditional Chinese medicine and Contour Projection‐stepwise regression offered an accurate, simple, low‐cost and eco‐friendly way for the rapid quantitative analysis of Shuanghuanlian oral liquid samples.     

Antiproliferation and Induction of Apoptosis in Ca9-22 Oral Cancer Cells by Ethanolic Extract of <em>Gracilaria tenuistipitata</em>

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.     

Quantitation of R-836, a New Oral Bronchodilator,by High Performance Liquid Chromatography using Manual or Robotic Sample Preparation

Abstract

A simple and selective high performance liquid chromatography (HPLC) method has been developed for the quantitation of R-836 (an investigational oral bronchodilator) in human plasma and urine, dog plasma and urine, and rat plasma. The method consists of reversed-phase HPLC with ultraviolet detection. Sample preparation involved a one-step protein precipitation procedure and was performed both manually and by a Zymate robotic system. Precision and accuracy results showed excellent reproducibility; results using the robotic procedure were slightly better than the manual procedure. The robotic procedure was capable of preparing the samples with minimal operator handling.     

Simultaneous determination of reduced and oxidized glutathione in freshly isolated rat hepatocytes and cardiomyocytes by HPLC with electrochemical detection

Glutathione and glutathione disulphide constitute an essential thiol redox system present in the cell. The balance in favour of the latter is an indication of oxidative stress. Glutathione and glutathione disulphide quantification in isolated cells may therefore be essential for the evaluation of mechanistic and comparative studies of toxic xenobiotics. In this study, a rapid and sensitive isocratic reverse-phase high-performance liquid chromatographic method using coulometric detection was implemented for the simultaneous detection of glutathione and glutathione disulphide, in freshly isolated hepatocytes and cardiomyocytes of the rat. The method implemented proved to be effective for the measurement of glutathione and glutathione disulphide in control conditions and for the detection of variations in this redox system, induced by tert-butylhydroperoxide. tert-Butylhydroperoxide is an organic peroxide, which has been used as a model molecule for inducing oxidative stress in isolated cells. A comparative study with a previously published HPLC-electrochemical detection method was performed.     

Aberrant proteins in the saliva of patients with oral squamous cell carcinoma

Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients’ 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients’ saliva α1‐antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α‐1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.     

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.     

Determination of nicorandil in plasma using high-performance liquid chromatography with photoconductivity and ultraviolet detection. Application to pre-clinical pharmacokinetics in beagle dogs

A sensitive and precise method for the determination of nicorandil, a new anti-anginal medication, is reported. The method involves solid-phase extraction of the drug and internal standard using Bond-Elut C18 extraction columns, reversed-phase high-performance liquid chromatography on a Zorbax-Phenyl column and detection with photoconductivity and ultraviolet detection in series. Photoconductivity, performed with the Tracor 965 photoconductivity detector, provided a limit of detection of 2 ng/ml in plasma (between-day coefficient of variation of 15%) but the linear range of response was limited to only about 100 ng/ml. Ultraviolet detection in series with the photoconductivity detector extended the linear range of the analytical system to 1000 ng/ml (coefficient of variation 4.4%). The utility of the method is demonstrated in a dog pharmacokinetic study in which a 5-mg intravenous dose was compared to a 10-mg oral solution dose in six beagle dogs. The oral solution was absorbed rapidly, achieving an average maximum concentration of 857 ng/ml in 11.2 min. The absolute bioavailability of nicorandil in dogs in this study was determined to be 84.2%.     

Fast Separation Method Development for Supercritical Fluid Chromatography Using an Autoblending Protocol

Supercritical fluid chromatography (SFC) is a powerful separation technique particularly in the area of enantioseparations. With rapid analysis speed, wide polarity compatibility, higher column efficiency and lower cost of the mobile phase, SFC is regarded as a better choice than high-performance liquid chromatography for drug discovery. In the development of separation method, the choice of modifier and/or additive is the key point of optimum separation. However, such screening of SFC is typically time-consuming. In this study, an autoblending protocol was introduced to speed up the modifier and/or additive screening process, which was performed on a separate programmable gradient proportioning system. The protocol prepares mobile phases on the fly to speed up the screening of modifiers and/or additives and reduces the waste of solutions. Furthermore, by switching mobile phase in the same run, separation of different types of compounds could also be achieved. This system was successfully applied to screen modifier–additive combinations of three alkaloids and three polyphenols by switching to two mobile phase conditions, as well as by a ternary additive mobile phase on an SFC system. The proposed protocol allows fast separation method development of SFC, which was proved to be rapid, simple, and reproducible.     

Rapid detection of Mycobacterium tuberculosis cells by using microtip-based immunoassay

This paper describes a microtip-based approach of concentrating target analytes for a highly sensitive bioassay. As an example, rapid screening of bacterial whole cells is presented to detect Mycobacterium tuberculosis (MTB), a pathogenic bacterium for human tuberculosis (TB). The concentration and detection is performed with three sequential steps of (1) attracting bacterial whole cells in the vicinity of a microtip by alternating current electroosmotic flow; (2) capturing the cells onto the microtip by capillary action; (3) binding fluorophore-labeled polyclonal antibodies to the cells followed by fluorescence measurement (immunofluorescence). Through this mechanism, MTB cells have been detected to the concentration of 8,000 cells/mL within 10 min. This sensitivity is comparable to that of Ziehl–Neelsen smear microscopy, a common culture-free screening method for diagnosis of TB. For comparison, Escherichia coli O157:H7 cells have also been detected to the concentration of 30,000 cells/mL in the same way.