A Novel Method for Relative Quantitation of N-Glycans by Isotopic Labeling Using 18O-Water

Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using ¹⁸O-water. The incorporation of the ¹⁸O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in ¹⁶O-water. A mathematical calculation method was also developed to determine the ¹⁸O/¹⁶O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility.     

Sensitive automated methods for phosphate and (Na+ plus K+)-ATPase

Sensitive automated methods for phosphate and (Na⁺ + K⁺)-ATPase are presented. The automated systems use sampler and pump modules from a Technicon autoanalyzer along with a Gilford spectrophotometer. The phosphate assay has a molar absorbance of approximately 87,000 m^?1 cm^?1 640 nm. The method uses a single color reagent and has been used successfully with perchloric acid digests of phospholipids.The (Na⁺ + K⁺)-ATPase method is based on the phosphate method. The method is comparable to manual methods in the amount of ATP and enzyme used per assay. The blank due to ATP is low. The precision of the assay on replicate samples is usually within ±1%. The total time delay for a single assay is less than 9 min and the method can be operated at a rate of 30 samples/hr. The method has been particularly useful in enzyme purification work. Procedures for the use of the method in kinetic studies are described     

Ion chromatographic study of sodium,potassium and ammonium in human body fluids with bulk acoustic wave detection

In the present paper, determination of Na⁺, K⁺ and NH₄⁺ in saliva and serum was carried out using an ion chromatography (IC) method with a bulk acoustic wave (BAW) sensor as detector and 2.0 mmol/l nitric acid as mobile phase. The IC–BAW method is simple, rapid and accurate. Comparison between the BAW detector and the conventional conductivity detector (CDD) was discussed. The IC–BAW showed agreement with the commonly used flame photometric method for Na⁺ and K⁺ and enzymatic method for NH₄⁺, as well as IC–CDD for those three cations.     

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples¹. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents^2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples^4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (³¹P-NMR) -a method that is expensive and requires specialized technical training⁶. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system⁷. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.     

Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (³⁵SO₄²⁻). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a ³⁵S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO₄²⁻) of 10⁻¹⁴ to 10⁻¹³ mol of SO₄²⁻ cell⁻¹ day⁻¹ were calculated, which is within the range of qSO₄²⁻ values previously reported for pure cultures of SRB (10⁻¹⁵ to 10⁻¹⁴ mol of SO₄²⁻ cell⁻¹ day⁻¹). qSO₄²⁻ values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10⁻¹⁰ to 7 × 10⁻¹⁰ mol of SO₄²⁻ cell⁻¹ day⁻¹), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.     

Gas chromatographic determination of total amino acids and protein in sediments using the ninhydrin-CO2 reaction

A sensitive modification of the ninhydrin-CO₂ method involving the gas chromatographic determination of the total protein and amino acid content of sediment is described. The method gives a linear response over the amino acid concentration range 10^?5 M to 4 × 10^?2 M. It can be used for whole sediment, hydrolysates and interstitial water. The performance of the method is compared with the fluorescamine method for primary amines.     

Determination of Δ-tetrahydrocannabinol levels in brain tissue using high-performance liquid chromatography with electrochemical detection

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the major psychoactive component of cannabis. To assist in investigating the mechanism(s) of action of Δ⁹-THC, a convenient method for determining its levels in brain tissue is required. We now describe a method for determining nanogram quantities of Δ⁹-THC in rat brain tissue. The method employs solvent extraction with methanol-hexane-ethyl acetate, followed by analysis using liquid chromatography with electrochemical detection. Overall recoveries were greater than 80%. The relationship between the peak-height ratio for processed standards extracted in the presence of tissue (Δ⁹-THC/internal standard) and the amount of Δ⁹-THC added was shown to be linear within the range of concentrations examined. Quantitative measurements of Δ⁹-THC in different brain regions following the intravenous administration of Δ⁹-THC are presented as examples of the applications of this method.     

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low‐cost, high‐throughput analytical methods for quality control. Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Results – Δ⁹‐THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r² = 0.9968. Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of Δ⁹‐THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars. Copyright © 2009 John Wiley & Sons, Ltd.     

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum)

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd²⁺) was developed for the first time for the wheat Triticum aestivum grown in Cd²⁺-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd²⁺. The ITCB-EDTA/Cd²⁺ complex was fixed with proteins in situ via the isothiocyano group. A new Cd²⁺-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd²⁺-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd²⁺-positive roots were compared with those of Cd²⁺-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd²⁺ content in plant tissues and illustrate Cd²⁺ translocation and cellular and subcellular responses of T. aestivum to Cd²⁺ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd²⁺ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd²⁺ in plant tissues and can be extended to other heavy metallic ions.     

A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.     

Double labeling of chromatin proteins,in vivo and in vitro,and their two-dimensional electrophoretic resolution

A modification of the two-dimensional electrophoretic method that involves nonequilibrium pH gradients has been adapted for high resolution of chromatin proteins from sea urchin embryos. A simple method of labeling the protein, in vitro, by reductive methylation with boro[³H]hydride to a specific activity of 100,000 cpm/μg of protein is detailed. Chromatin protein may be labeled, in vivo with ¹⁴C-amino acids, and newly synthesized (³H and ¹⁴C-labeled) and preexistent proteins (only ³H labeled) may be distinguished. The method reveals that sea urchin embryo chromatin contains over 200 proteins.     

Separation of double-label liquid scintillation samples by half-life

A method is presented for separation of double- or triple-labeled liquid scintillation samples based on the partial decay of one of the nuclides. Validity of the procedure has been demonstrated with the following combinations of nuclides commonly encountered in laboratory situations; ¹³¹I-¹⁴C, ³²P-¹⁴C, and ²⁰³Hg-¹⁴C, Separation of all nuclide pairs agrees favorably with predieted values except when the activity ratios exceed 10³. The determinations are independent of quenching and nuclide spectra and greatly augment the number of β-β and β-γ combinations resolved by liquid scintillation spectrometry. The utility of this method for separation of triple-labeled samples is discussed.     

Measurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method

A method for the measurement of the cytosolic Na⁺ concentration in intact synaptosomes is described. This method makes use of a pH sensitive dye (BCECF) that can be loaded into the cytosol and a relatively specific ionophore (monensin) that can exchange Na⁺ for H⁺ across the synaptosomal membrane. By setting conditions such that there is no electrochemical potential difference for H⁺ across the membrane (no membrane potential and pH_i = pH_o), addition of ionophore would induce a H⁺ flux only if there is a concentration difference for Na⁺. Thus, when there is no fluorescence change (no cytosolic pH change) extracellular [Na⁺] equals intrasynaptosomal [Na⁺. The intrasynaptosomal [Na⁺] concentration was determined to be 7 ± 3 mM (n = 5; mean ± S.E.). The results obtained with this fluorescence method are compared with estimates obtained by atomic absorption spectrometry. Limitations and applications of the method are discussed.     

Determination of isotope distribution in labeled lysine

A method is described for the degradation of ¹⁴C-labeled lysine with determination of radioactivity in the individual C atoms. The method depends on permanganate oxidation of lysine to give δ-aminopentanoic acid, γ-aminobutyric acid, β-alanine, and glycine, and on ninhydrin decarboxylation of glycine. The method will be equally useful for determining the distribution of ¹⁵N and ³H in lysine.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

Application of the change in partition coefficient with pH to the structure determination of alkyl substituted guanosines.

A versatile method for the determination of the ionization of guanosine is described. Suitably derivatized alkyl-guanosines are partitioned between organic solvents and aqueous buffer solutions at various pH values. Ionization is revealed by a change in partition coefficient with pH. The method is ideally suited for application to micro samples since any quantitative method can be used to determine the partition coefficient. A procedure for distinguishing N¹ or O⁶, N², and C⁸ alkylation of guanosine is described.     

Efficient fluorography of 3H and 14C on thin layers.

Three methods for efficient detection of ³H and ¹⁴C on thin layers are described. The first method uses 0.4% PPO dissolved in 2-methylnaphthalene. It is approximately 15-fold more efficient than 7% PPO-ether for ³H detection and 15-fold more efficient than autoradiography for ¹⁴C (or ³⁵S) detection. The second method uses PPO dissolved in ether. Efficiency of ³H detection increases as the PPO concentration is increased. The third method uses melted PPO and is as efficient as the first method when the film is exposed at ?78°C.     

Crystal violet–G-quadruplex complexes as fluorescent sensors for homogeneous detection of potassium ion

A novel K⁺ detection method was reported using a label-free G-quadruplex-forming oligonucleotide and a triphenylmethane fluorescent dye crystal violet (CV). This method is based on the fluorescence difference of some CV/G-quadruplex complexes in the presence of K⁺ or Na⁺, and the fluorescence change with the variation of K⁺ concentration. According to the nature of the fluorescence change of CV as a function of ionic conditions, two K⁺ detection modes can be developed. One is a fluorescence-decreasing mode, in which T₃TT₃ (5′-GGGTTTGGGTGGGTTTGGG) is used, and the fluorescence of CV decreases with an increased concentration of K⁺. The other is a fluorescence-increasing mode, in which Hum21 (5′-GGGTTAGGGTTAGGGTTAGGG) is used, and the fluorescence of CV increases with an increased concentration of K⁺. Compared with some published K⁺ detection methods, this method has some important characteristics, such as lower cost of the test, higher concentrations of Na⁺ that can be tolerated, adjustable linear detection range and longer excitation and emission wavelengths. Preliminary results demonstrated that the method might be used in biological systems, for example in urine.     

Application and efficiency of scintillation autoradiography for Drosophila polytene chromosomes

Summary A rapid method of autoradiography using the scintillation cocktail (Toluene and scintillation fluid, Omnifluor) has been described earlier. Its application and efficiency have been tested using both ³H-thymidine and ³H-uridine. The optimum time required for processing the autoradiograms has been found to be 24 h dry exposure followed by 48 h in the scintillation mixture. Detailed analysis of the autoradiograms with ³H-uridine reveals that with the rapid method the 100% level of labelling index is reached by 48 h while with the conventional method the same level is reached by 10 to 12 days of dry exposure. The maximum grain density is reached by 16 to 17 days by the conventional method. While by the rapid method, the maximum grain density is approximately 80% of the control, this grain density is reached by 48 h (plus 24 h of dry exposure) and thereafter forms a plateau. With Toluene alone the grain density never exceeds 20%. The background is also relatively low and less variable in the O-T-processed autoradiograms, as compared to the two controls. These results support that the scintillation fluid plays the key role in augmenting the labelling. Furthermore, although the maximum grain density by the rapid technique is 80% of the control, the grain density obtained by the rapid method gives less coincidence and superimposition of grains.On the other hand, with ³H-thymidine, although all labelling patterns could be resolved, the labelling index (i.e., percent of labelled cells) is about 40% at 48 h (plus 24 h) and about 79.5% at 96 h with the rapid method, as compared to about 30% and 44% with the conventional method at the two time points, respectively. Only with 16–17 days' dry exposure the ³H-thymidine labelling index increases to 67%. The frequency of the initial patterns (DD-2C) which are usually less frequent, has been found to have increased with the rapid method. No difference in grain density of labelling of ³H-thymidine could be detected between the rapid method and the conventional method. The resolution of grains also seems to be better by the rapid method, due probably to smaller size and lack of superimposition of grains. Other applications, advantages and limitations have been discussed.     

Measurement of CO(2) Assimilation in Soils: an Experiment for the Biological Exploration of Mars

A method is described for the measurement of ¹⁴CO₂ assimilation by microorganisms in soils. A determination involves exposing soil to ¹⁴CO₂, pyrolyzing the exposed soil, trapping the organic pyrolysis products on a column of firebrick coated with CuO, combusting the trapped organics by heating, and measuring the radioactivity in the CO₂ produced in the combustion. The detection of significant levels of ¹⁴C in the trapped organic fraction appears to be an unambiguous indication of biological activity. The ¹⁴CO₂ which is adsorbed or exchanged into soils by nonbiological processes does not interfere. The method easily detects the ¹⁴CO₂ fixed by 10² to 10³ algae after light exposure for 3 to 24 hr. Assimilation of ¹⁴C is also demonstrable in dark-exposed soils containing 10⁵ to 10⁶ heterotrophic bacteria. Possible applications of the method in the biological exploration of Mars are discussed.