Quantitative autoradiography in boron neutron capture therapy considering the particle ranges in the samples

PurposeBoron neutron capture therapy is a cellular-scale particle therapy exploiting boron neutron capture reactions in boron compounds distributed in tumour cells. Its therapeutic effect depends on both the accumulation of boron in tumour cells and the neutron fluence. Autoradiography is used to visualise the micro-distribution of boron compounds.MethodsHere, we present an equation for the relationship between boron concentration and pit density on the solid-state nuclear track detector, taking into consideration the particle ranges in the samples. This equation is validated using liver-tissue sections and boron standard solutions. Moreover, we present a simple co-localisation system for pit and tissue-section images that requires no special equipment.ResultsThe equation reproduces the experimentally observed trends between boron concentration and pit density. This equation provides a theoretical explanation for the widely used calibration curve between pit density and boron concentration; it also provides a method to correct for differences of tissue-section thickness in quantitative autoradiography.ConclusionsUsing the equation together with this co-localisation system could improve micro-scale quantitative estimation in tissue sections.     

Spectrophotometric method for high biomass concentration measurements

Continuous monitoring of concentration is important in the optimal control of bioreactors. Spectrophotometry provides a simple, accurate, and rapid way for measuring cell concentration of unicellular microorganisms, based on either Beer's law or calibration curves prepared using standard solutions. However, this method is limited to a low range of microbial concentrations, because Beer's law deviates significantly at high concentrations. In the present investigation, based on experimental work, a new technique is posed to monitor the concentration of microbial cells as high as 100 g DW/L. this is achieved by using a mixture of known concentration as reference, rather than the "ideal blank" with zero concentration of analyte. As a result, a new equation is developed that, although applied here only to microbial concentration, in principle can be used for monitoring the concentration of any optically sensitive material.     

A neutron activation technique for manganese measurements in humans

Manganese (Mn) is an essential element for humans, animals, and plants and is required for growth, development, and maintenance of health. Studies show that Mn metabolism is similar to that of iron, therefore, increased Mn levels in humans could interfere with the absorption of dietary iron leading to anemia. Also, excess exposure to Mn dust, leads to nervous system disorders similar to Parkinson's disease. Higher exposure to Mn is essentially related to industrial pollution. Thus, there is a benefit in developing a clean non-invasive technique for monitoring such increased levels of Mn in order to understand the risk of disease and development of appropriate treatments.To this end, the feasibility of Mn measurements with their minimum detection limits (MDL) has been reported earlier from the McMaster group. This work presents improvement to Mn assessment using an upgraded system and optimized times of irradiation and counting for induced gamma activity of Mn. The technique utilizes the high proton current Tandetron accelerator producing neutrons via the ⁷Li(p,n)⁷Be reaction at McMaster University and an array of nine NaI (Tl) detectors in a 4π geometry for delayed counting of gamma rays. The neutron irradiation of a set of phantoms was performed with protocols having different proton energy, current and time of irradiation. The improved MDLs estimated using the upgraded set up and constrained timings are reported as 0.67 μgMn/gCa for 2.3 MeV protons and 0.71 μgMn/gCa for 2.0 MeV protons. These are a factor of about 2.3 times better than previous measurements done at McMaster University using the in vivo set-up. Also, because of lower dose-equivalent and a relatively close MDL, the combination of: 2.0 MeV; 300 μA; 3 min protocol is recommended as compared to 2.3 MeV; 400 μA; 45 s protocol for further measurements of Mn in vivo.     

An evaluation of thermal and epithermal neutron activation analysis compton suppression methods for biological reference materials

For neutron activation analysis (NAA), the usual matrix problems of sodium, chlorine, and bromine are well known to give rise to high backgrounds that inhibit the determination of several trace elements for short-lived or medium-lived NAA. For long counting times in long-lived NAA, very low backgrounds are required to achieve good sensitivities. We have investigated the use of thermal and epithermal NAA in conjunction with Compton suppression to determine several elements such as arsenic, antimony, cadmium, and mercury, at the level of a few nanograms. The values of these techniques are discussed in contrast to the standard radiochemical methods.     

Factors controlling equilibrium boron (B) concentration in nutrient solution buffered with B-specific resin (Amberlite IRA-743)

In conventional solution culture where boron (B) is added as boric acid, fluctuating external B supply often produces confounding and ill-defined physiological and biochemical responses in plants, especially when grown at deficient and marginal B supply. Our previous studies proposed the use of the B-specific resin – Amberlite IRA-743 to develop a B-buffered solution culture. The present study aims to evaluate crucial factors determining equilibrium B concentrations in nutrient solution buffered with the B-loaded resin, including the B loading of the resin, pH in the nutrient solution and B removal from the solution. The equilibrium B concentrations in nutrient solution were determined by both the amount of B sorbed by the resin and the solution pH. At pH 6.05±0.05, the relationship between the resin B content and equilibrium B concentration in the nutrient solution is closely described by the equation: Y = 18.8 X^1.457 [ where, Y = equilibrium B concentration (μM) in nutrient solution and X = B content of the resin (mg B g⁻¹ moist resin)]. However, at a given resin B content, lowering solution pH from 7 to 4 significantly increased B concentrations in solution through the release of B from the solid phase of the resin beads. The B-loaded resin was capable of maintaining stable B concentrations in the nutrient solutions, ranging from deficient to marginally adequate B concentrations for dicot species. In conclusion, B concentrations ranging from 0.05 to 11 μM, were buffered for 5 days with the resin loaded with 0.004 – 0.691 mg B g⁻¹ moist resin in the nutrient solution. Precise pH control in the nutrient solution is critical for the success of a B-buffered solution culture study. This revised version was published online in June 2006 with corrections to the Cover Date.     

An assay for thiaminase I in complex biological samples

An alternative method for measuring thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, resulting in a large decrease in absorbance at 411nm. This new assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high-throughput analysis in a 96-well format with complex biological samples.     

An improved and easy technique for polyamine determination in biological samples : Application to cell-free system from hypertrophied rat heart

An accurate, improved cation-exchange chromatographic method using o-phthalaldehyde and ultraviolet detection at 280 nm for the determination of free polyamines (putrescine, spermidine, spermine) has been developed. Different samples, such as the 105,000 g supernatant of reticulocyte or heart muscle, and KCl ribosomal wash containing initiation factors, can be analysed. The minor modification of reagents results in a good precision and sensitivity, which is demonstrated by a relative standard deviation of 5–9% and recoveries of 98%. This technique is of particular interest because it allows polyamine determination in biological samples with high concentrations of salt.     

An adaptable HPLC method for the analysis of frequently used antibiotics in ocular samples

Four different antibiotics, delivered individually to rabbit eyes via hydrophilic intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH 5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to 200 pg. The overall accuracy of the method was ≥90%. With minor modifications, the method was optimized for each of the agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular penetration of these drugs.     

An ultrasensitive gold nanoparticles improved real-time immuno-PCR assay for detecting diethyl phthalate in foodstuff samples

A specific polyclonal antibody targeting diethyl phthalate (DEP) with the higher antibody titer at 1:120,000 has been obtained, and an ultrasensitive and high-throughput direct competitive gold nanoparticles improved real-time immuno-PCR (GNP–rt–IPCR) technique has been developed for detecting DEP in foodstuff samples. Under optimal conditions, a rather low linearity is achieved within a range of 4 pg L⁻¹ to 40 ng L⁻¹, and the limit of detection (LOD) is 1.06 pg L⁻¹. Otherwise, the GNP–rt–IPCR technique is highly selective, with low cross-reactivity values for DEP analogs (<5%). Finally, the concentrations of DEP in foodstuff samples by the GNP–rt–IPCR method range from 0.48 to 41.88 μg kg⁻¹. Satisfactory recoveries (88.39–112.79%) and coefficient of variation values (8.38–12.77%) are obtained. The consistency between the results obtained from GNP–rt–IPCR and gas chromatography–mass spectrometry (GC–MS) is 98.3%, which further proves that GNP–rt–IPCR is an accurate, reliable, rapid, ultrasensitive, and high-throughput method for batch determination of trace amounts of DEP in foodstuff samples.     

An enzymatic method of analysis for GDP-L-fucose in biological samples,involving high-performance liquid chromatography

To investigate the biological significance of GDP-L-fucose, we established a unique method for the determination of GDP-L-fucose levels in microsomal fractions, using an HPLC assay of alpha 1-6-fucosyltransferase (alpha1-6-FucT), an enzyme that catalyzes the synthesis of core fucosylation in N-glycans. A microsomal protein and a large excess of fluorescence-labeled synthetic oligosaccharide (a substrate) were incubated with a large excess of alpha1-6-FucT. The fluorescent intensity of the fucosylated reaction product, which was analyzed by isocratic reverse phase HPLC, was proportional to the level of GDP-L-fucose in the microsomal fractions over the range 0.20-10 pmol. This assay is applicable to the determination of the GDP-L-fucose content in various cancer cell lines as well as rat liver and would be useful in developing a better understanding of the fucosylation potential of such cells and tissues.     

An automated approach for fringe frequency estimation and removal in infrared spectroscopy and hyperspectral imaging of biological samples

In infrared spectroscopy of thin film samples, interference introduces distortions in spectra, commonly referred to as fringes. Fringes may alter absorbance peak ratios, which hampers the spectral analysis. We have previously introduced extended multiplicative signal correction (EMSC) for fringes correction. In the current article, we provide a robust open-source algorithm for fringe correction in infrared spectroscopy and propose several improvements to the Fringe EMSC model. The suggested algorithm achieves a more precise fringe frequency estimation by mean centering of the measured spectrum and applying a window function prior to the Fourier transform. It selects two frequencies from a user defined number of maxima in the Fourier domain. The improved Fringe EMSC algorithm is validated on two experimental datasets, one of them being a hyperspectral image. Techniques for separating sample spectra from background spectra in hyperspectral images, and techniques to identify spectra affected by fringes are also provided.     

The Monte Carlo simulation of the biological effect of the 10B(n, alpha)7Li reaction in cells and tissue and its implication for boron neutron capture therapy

The energy deposition in the nucleus of cells exposed to the 10B(n, alpha)7Li neutron capture reaction has been calculated and compared to the measured biological effect of this reaction. It was found that a considerable distribution of hit sizes to the nucleus occurs. The comparison of hit size frequency with the observed survival indicates that not every hit, independent of its size, can lead to cell death. This implies the existence of a hit size effectiveness function. The analysis shows that the location of boron relative to the radiation-sensitive volume of the cell is of great importance and that average dose values alone are of limited use for predicting the biological effect of this reaction. Boron accumulating in the cell nucleus is much more efficient in cell killing than the same amount of boron uniformly distributed; its presence in one cell, however, has little effect on its neighboring cells in a tissue. When boron is present on the cell surface of a tissue (as presumably delivered by antibodies), its cell-killing effect is greatly reduced compared to that in uniform distribution. However, in this case much of the dose to one cell comes from neutron capture reactions occurring on the surface of its neighbor cells. These data have implications for the choice of boron carries in neutron capture therapy. The mathematical analysis carried out here is similar to that proposed recently for low-level exposure effects of radiation, taking mutation and/or carcinogenesis as biological effects. The results here show that high-level exposure to high-LET particles (resulting in cell killing) should be treated in an analogous manner.     

A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO₂) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP₆ (phytate), its pyrophosphate derivatives InsP₇ and InsP₈, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP₆, InsP₇ and InsP₈ were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO₂ bead purification also enabled us to quantify InsP₆ in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP₆ phosphatase in human plasma, and secondly, InsP₆ is undetectable in either fluid. These observations seriously question reports that InsP₆ is present in human biofluids and the advisability of using InsP₆ as a dietary supplement.     

An improved LC-MS/MS method for the quantification of prostaglandins E(2) and D(2) production in biological fluids

We report an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that accurately measures prostaglandins D(2) (PGD(2)) and E(2) (PGE(2)) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/ml (0.20 pg, 0.55 fmol on-column), and the interday and intraday coefficients of variation were less than 5%. Both d(4)-PGE(2) and d(4)-PGD(2) were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8h to measure PGD(2) accurately, whereas preparation time did not affect PGE(2) measurement due to its greater stability in biological samples. As an application of the method, PGD(2) and PGE(2) were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE(2) but no PGD(2), whereas the murine macrophage cell line RAW 264.7 produced PGD(2) and only trace amounts of PGE(2). This direct comparison showed that COX-2 gene expression can lead to differential production of PGD(2) and PGE(2) by epithelial cells and macrophages. Because PGE(2) is antiasthmatic and PGD(2) is proasthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macrophages in the lung contributes to the pathogenesis of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and lung cancer.