Boron concentration measurement in biological tissues by charged particle spectrometry

Measurement of boron concentration in biological tissues is a fundamental aspect of boron neutron capture therapy, because the outcome of the therapy depends on the distribution of boron at a cellular level, besides on its overall concentration. This work describes a measurement technique based on the spectroscopy of the charged particles emitted in the reaction ¹⁰B(n,α)⁷Li induced by thermal neutrons, allowing for a quantitative determination of the boron concentration in the different components that may be simultaneously present in a tissue sample, such as healthy cells, tumor cells and necrotic cells. Thin sections of tissue containing ¹⁰B are cut at low temperatures and irradiated under vacuum in a thermal neutron field. The charged particles arising from the sample during the irradiation are collected by a thin silicon detector, and their spectrum is used to determine boron concentration through relatively easy calculations. The advantages and disadvantages of this technique are here described, and validation of the method using tissue standards with known boron concentrations is presented.     

Methods to measure the intracellular concentration of unlabeled compounds within cultured cells using liquid chromatography/tandem mass spectrometry

A high-throughput and sensitive liquid chromatography/tandem mass spectrometry assay was established to detect total unlabeled hepatitis C virus inhibitor concentrations in replicon cells. The intracellular concentrations determined by this assay correlated well with concentrations obtained using radiolabeled compound. Some compounds accumulated inside the cells, with concentrations up to 300-fold higher than the input concentration. Confocal microscopic evaluation of two fluorescent-tagged inhibitors confirmed high accumulation inside the cells, sequestered inside vesicles within the cytoplasm. Incubation of cells with compound at 4 °C revealed that nonspecific binding to the outside of the cell membrane and to the cell culture plate occurred for some compounds. Therefore, the total concentration of compound extracted at 37 °C was reduced by the amount that was nonspecifically bound at 4 °C to yield the amount of compound inside the cells. A modification of the protocol was used for compounds with low intracellular concentrations in which cells were harvested with trypsin-EDTA prior to extraction. This eliminated the nonspecific binding to the cell culture plate and decreased the overall background of the assay. This assay was used to understand differences in cellular potency between compounds and the effects of serum proteins on the metabolic stability of compounds during incubation with cells.     

An improved neutron autoradiography set-up for 10B concentration measurements in biological samples

Aim

Boron Neutron Capture Therapy (BNCT) is a binary hadrontherapy which exploits the neutron capture reaction in boron, together with a selective uptake of boronated substances by the neoplastic tissue. There is increasing evidence that future improvements in clinical BNCT will be triggered by the discovery of new boronated compounds, with higher selectivity for the tumor with respect to clinically used sodium borocaptate (BSH) and boronophenylalanine (BPA).

Background

Therefore, a ¹⁰B quantification technique for biological samples is needed in order to evaluate the performance of new boronated formulations.

Materials and methods

This article describes an improved neutron autoradiography set-up employing radiation sensitive films where the latent tracks are made visible by proper etching conditions.

Results

Calibration curves for both liquid and tissue samples were obtained.

Conclusions

The obtained calibration curves were adopted to set-up a mechanism to point out boron concentration in the whole sample.     

A MR imaging procedure to measure tarsal bone rotations

Magnetic resonance imaging offers unique insights into three-dimensional foot bone motion. Thereby, adequate devices enabling defined loading and positioning of the foot are needed to profit from this noninvasive procedure. Tarsal bone positions of three healthy subjects were repeatedly measured in a pronated and a supinated foot excursion under bodyweight with a newly developed MR imaging procedure. The quantification of the transferred motion from the loading and positioning device to the calcaneus and an estimation of the required degrees to distinguish between tarsal joint rotations were used to evaluate the applicability of the procedure to investigate tarsal joint motion. It was found that 45-70% (75-95%) of the externally applied 15 deg foot pronation (supination) were transferred to the calcaneus. Furthermore, the talonavicular joint showed the largest amount of rotation up to 20 deg eversion-inversion and abadduction, followed by the subtalar joint showing nearly half of that motion. Considerably less motion was found between the cuboid and calcaneus (about 2-6 deg) and the cuboid nearly did not rotate relative to the navicular (on average 1 deg). The estimated necessary differences between tarsal joint movements to identify individual kinematic behavior were in the order of 2 deg (4 deg related to the talonavicular joint). Since the results were in agreement with the literature, it is concluded that the applicability of the presented procedure to investigate tarsal bone mechanics is warranted. The possibility to evaluate 3D tarsal joint motion in combination with bone morphology (e.g., joint curvature) may provide new insights in the still uncertain relationship between foot function and foot morphology.     

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.     

Accuracy of a CT-based bone contour registration method to measure relative bone motions in the hindfoot

For measuring the in-vivo range of motion of the hindfoot, a CT-based bone contour registration method (CT-BCM) was developed to determine the three-dimensional position and orientation of bones. To validate this technique, we hypothesized that the range of motion in the hindfoot is equally, accurately measured by roentgen stereophotogrammetric analysis (RSA) as by the CT-BCM technique.Tantalum bone markers were placed in the distal tibia, talus and calcaneus of one cadaver specimen. With a fixed lower leg, the cadaveric foot was held in neutral and subsequently loaded in eight extreme positions. Immediately after acquiring a CT-scan with the foot in a position, RSA radiographs were made. Bone contour registration and RSA was performed. Helical axis parameters were calculated for talocrural and subtalar joint motion from neutral to extreme positions and between opposite extreme positions. Differences between CT-BCM and RSA were calculated.Compared with RSA, the CT-BCM data registered an overall root mean square difference (RMSd) of 0.21° for rotation about the helical axis, and 0.20 mm translation along the helical axis for the talocrural and subtalar joint and for all motions combined. The RMSd of the position and direction of the helical axes was 3.3 mm and 2.4°, respectively. The latter errors were larger with smaller helical rotations.The differences are similar to those reported for validated RSA and thus are not clinically relevant. Concluding, CT-BCM is an accurate and accessible alternative for studying joint motion, as it does not have the risk of infection and overlapping bone markers.     

A pharmacokinetic model for the concentration of 10B in blood after boronophenylalanine-fructose administration in humans

An open two-compartment model has been developed for predicting (10)B concentrations in blood after intravenous infusion of the l-p-boronophenylalanine-fructose complex (BPA-F) in humans and derived from studies of pharmacokinetics in 24 patients in the Harvard-MIT Phase I clinical trials of BNCT. The (10)B concentration profile in blood exhibits a characteristic rise during the infusion to a peak of approximately 32 microg/g (for infusion of 350 mg/kg over 90 min) followed by a biphasic exponential clearance profile with half-lives of 0.34 +/- 0.12 and 9.0 +/- 2.7 h, due to redistribution and primarily renal elimination, respectively. The model rate constants k(1), k(2) and k(3) are 0.0227 +/- 0.0064, 0.0099 +/- 0.0027 and 0.0052 +/- 0.0016 min(-1), respectively, and the central compartment volume of distribution, V(1), is 0.235 +/- 0.042 kg/kg. The validity of this model was demonstrated by successfully predicting the average pharmacokinetic response for a cohort of patients who were administered BPA-F using an infusion schedule different from those used to derive the parameters of the model. Furthermore, the mean parameters of the model do not differ for cohorts of patients infused using different schedules.     

Gas chromatography/mass spectrometry method to quantify blood hydroxycitrate concentration

Hydroxycitrate (HCA), a popular dietary supplement for weight loss, is a competitive inhibitor of ATP-citrate lyase, an extramitochondrial enzyme involved in the initial steps of de novo lipogenesis (DNL). Although animal studies have shown that HCA effectively inhibits DNL and induces weight loss, these findings have not been consistent in humans. This raises the possibility that the bioavailability of HCA may differ among species. We developed a new GC/MS method to measure HCA levels in blood, using [U-(13)C]citrate (CA*) as internal standard to account for losses associated with the isolation, derivatization, and measurement of HCA. HCA and CA* were derivatized with BSTFA + 10% TMCS and analyzed using PCI/GC/MS (CA*, m/z 471; and HCA, m/z 553). The plasma HCA concentration was measured over a 3.5-h period in four subjects having ingested 2 g of HCA. Their plasma HCA concentration ranged from 0.8 to 8.4 microg/ml 30 min and 2 h after ingestion, respectively. These results demonstrate that when taken acutely, HCA is absorbed, yet present in small quantities in human plasma. This simple method requiring minimal sample preparation is able to measure trace amounts of HCA with accuracy and precision.     

A rapid and sensitive bioassay to measure bone morphogenetic protein activity

Background  

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily and were originally identified as proteins that induce ectopic bone formation. BMPs were shown subsequently to be involved in several biological processes during development and in adult tissues through the regulation of the growth, differentiation and apoptosis of various cell types. An alkaline phosphatase (ALP)-based assay is the most widely used assay to evaluate BMP activity. However, the ALP assay is not rapid and not sensitive enough to measure BMP activity at physiological concentrations. In this paper, we describe a highly sensitive, rapid, and specific cell-based assay for the quantification of BMP activity.     

An application of nanoindentation technique to measure bone tissue Lamellae properties

Measuring the microscopic mechanical properties of bone tissue is important in support of understanding the etiology and pathogenesis of many bone diseases. Knowledge about these properties provides a context for estimating the local mechanical environment of bone related cells thait coordinate the adaptation to loads experienced at the whole organ level. The objective of this study was to determine the effects of experimental testing parameters on nanoindentation measures of lamellar-level bone mechanical properties. Specifically, we examined the effect of specimen preparation condition, indentation depth, repetitive loading, time delay, and displacement rate. The nanoindentation experiments produced measures of lamellar elastic moduli for human cortical bone (average value of 17.7 +/- 4.0 GPa for osteons and 19.3 +/- 4.7 GPa for interstitial bone tissue). In addition, the hardness measurements produced results consistent with data in the literature (average 0.52 +/- 0.15 GPa for osteons and 0.59 +/- 0.20 GPa for interstitial bone tissue). Consistent modulus values can be obtained from a 500-nm-deep indent. The results also indicated that the moduli and hardnesses of the dry specimens are significantly greater (22.6% and 56.9%, respectively) than those of the wet and wet and embedded specimens. The latter two groups were not different. The moduli obtained at a 5-nm/s loading rate were significantly lower than the values at the 10- and 20-nm/s loading rates while the 10- and 20-nm/s rates were not significantly different. The hardness measurements showed similar rate-dependent results. The preliminary results indicated that interstitial bone tissue has significantly higher modulus and hardness than osteonal bone tissue. In addition, a significant correlation between hardness and elastic modulus was observed.     

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.     

Application of high-performance liquid chromatography-electrospray ionization-mass spectrometry to measure microsomal membrane transport of glucuronides

A primary reason for poor characterization of microsomal transport to date is the limitations of the measurement techniques used. Radiodetection provides sufficient sensitivity, but it can be applied only when labeled analogue is available. In this article, we report the novel application of high-performance liquid chromatography and electrospray tandem mass spectrometry (LC-MS/MS) in "rapid filtration" transport assays. The method was developed using glucuronides, but it is adaptable to any compound that can be measured with LC-MS/MS. Because of the high sensitivity and accuracy of this detection technique, the substrates can be used at their physiological concentration in the experiments. The new methodology does not require radiolabeling, so it remarkably widens the range of possible substrates to investigate and allows simultaneous detection as well as monitoring of substrate stability during the experiments.     

Posttranslational modifications to human bone sialoprotein determined by mass spectrometry.

Bone sialoprotein (BSP) is an acidic 301 amino acid protein expressed by osteoblasts and at a low level by hypertrophic chondrocytes. Its expression is highest during early stages of bone formation, and it is particularly abundant in the cells lining the surface of newly formed trabeculae. BSP contains numerous substituents which are anionic in nature and apparently essential for the function of the protein. Thus, the proposed role of BSP in hydroxyapatite nucleation and growth may depend on such modifying groups. The posttranslational modifications include several acidic oligosaccharides as well as phosphate and sulfate groups. This work combines matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with selective enzyme treatment of BSP to provide new information on the precise distribution and structure of oligosaccharides, sulfate, and phosphate groups in BSP isolated from human bone. The results provide a high level of detail in the location of these modifying groups toward the end of providing a basis for further understanding the function of BSP in bone nucleation.     

Single particle electron microscopy in combination with mass spectrometry to investigate novel complexes of membrane proteins

Large data sets of molecular projections of the membrane proteins Photosystem I and Photosystem II from cyanobacteria were analyzed by single particle electron microscopy (EM). Analysis resulted in the averaging of 2D projections from the purified complexes but also in the simultaneous detection and averaging of 2D projections from large contaminating complexes, which were present in frequencies as low as 0.1%. Among them T-shaped and L-shaped contaminants were found. The L-shaped particles could be assigned to Complex I just from the shape, although no Complex I from a cyanobacterium has been structurally characterized. A systematic comparison by single particle EM and mass spectrometry of two differently purified Photosystem II complexes resulted in the assignment of PsbZ, a small peripheral subunit of 6.8kDa, within the structure. Together these data suggest that screening for membrane protein structures by single particle EM and mass spectrometry may be a new approach to find novel structures of such proteins. We propose here a scheme for searching for novel membrane protein structures in specific types of membranes. In this approach single particle EM and mass spectrometry, after pre-fractionation using one- or multidimensional protein separation techniques, are applied to characterize all its larger components.     

A high-throughput competitive scintillation proximity aminoacyl-tRNA synthetase charging assay to measure amino acid concentration

An enhanced method to measure the concentration of individual naturally occurring free amino acids in solution is described. This relatively simple but robust method combines two previously reported procedures: the use of scintillation proximity assay (SPA) technology to measure aminoacyl-tRNA synthetase (aaRS) activity and the use of aaRS activity to measure amino acid concentration using the enzymatic isotope dilution technique. The format described is called an aaRS competitive scintillation proximity assay (cSPA). This cSPA takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same nonradiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid. Under equilibrium conditions, in the case of limiting tRNA, the rate of the enzyme-catalyzed reaction relative to substrate concentration becomes irrelevant and the enzymatic isotopic dilution technique becomes the simple isotopic dilution technique. Due to the exquisite specificity of the reaction, a crude mixture of tRNAs and aaRSs can be used to detect the concentration of a particular amino acid without interference from noncognate amino acids. When used to monitor aminopeptidase M activity, this assay produced similar results in time course and inhibition experiments as compared with a traditional fluorescent assay. High-throughput compatibility was demonstrated by screening 12,000 compounds against aminopeptidase M in 384-well microtiter plates with Z factors ranging from 0.53 to 0.70. This competitive assay can be used as a general method to detect amino acids at concentrations less than 100 nM and to monitor enzyme activity in biological samples, and it is amenable to high-throughput screening.     

The design and development of a biosensor to measure the concentration of meconium in amniotic fluid

Meconium aspiration syndrome occurs in 0.2% to 1% of all deliveries and has a mortality rate as high as 18%. The disease is responsible for 2% of all perinatal deaths. Meconium may be classified as being thick or thin, but this assessment is normally performed visually by clinicians. A "meconiumcrit" analysis has been developed to objectively define the concentration of meconium. However, this analysis does not provide real-time continuous readings. This study focused on the design and development of a sensor to provide an objective, continuous, real-time assessment of meconium thickness. Meconium has an absorption spectrum centered at 410 nm and observes Beer's law. Blue light centered at 430 nm was delivered through meconium solutions, and a photodiode translated the strength of the incoming light into a voltage. This voltage was analyzed by a microcontroller to determine the concentration of meconium.     

Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila

The role of hydrogen peroxide (H(2)O(2)) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H(2)O(2) in vivo. Here we describe a method for assessing changes in H(2)O(2) within the mitochondrial matrix of living Drosophila. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H(2)O(2) to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H(2)O(2). To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography-tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H(2)O(2) in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H(2)O(2) production in mouse and cell culture studies.     

Standardization of the antibody to hepatitis B surface antigen concentration

We examine sources of potential bias in the estimation of antibody to hepatitis B surface antigen concentrations by a calibration curve for conversion of RIA units to international units. We show by calculation and example that very large biases may exist, whereas accurate estimation is needed in screening programmes and in clinical trials for the evaluation of the immunogenicity of various types and schedules of hepatitis B vaccine. It is recommended that the danger of large biases be avoided by using the laboratory's own calibration curve, calibrated against dilutions of the WHO standard, using a standard as positive control in the radioimmunoassay. Furthermore, serum samples should be diluted to a concentration close to that of the positive control.