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.     

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.     

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.