Rapid, high-throughput method for the quantification of thyroid hormones in rat blood serum using isotope-dilution LC–MS/MS

Aim: Numerous guideline studies required for regulatory toxicity testing now include the measurement of the thyroid hormones 3,3′,5-triiodo-L-thyronine (T3) and L-thyroxine (T4) in blood serum from rodents. A rapid, high-throughput method for the determination of the thyroid hormones T4 and T3 is reported. Materials & methods: Sample preparation is done using a 96-well microtiter plate format. Stable isotope analogs of both hormones are used as internal standards for study and quality control samples. Results & conclusion: The validated quantification levels are T3: 10 pg/ml and T4: 1 ng/ml, with CVs of <10% at the limit of quantification and up to 50*limit of quantification. The use of isotope analog internal standards eliminates the need for quantitative transfers and complete evaporations.

An amended potassium persulfate ABTS antioxidant assay used for medicinal plant extracts revealed variable antioxidant capacity based upon plant extraction process

Use of potassium persulfate (K2S208) for oxidation of 7.0 mM ABTS to a stable ABTS radical for antioxidant studies was first reported in 1999. A feature of this popular antioxidant assay has been the requirement of an overnight reaction (6 to 12 h) for the formation of a stable ABTS colored radical. It is now reported that when the concentration of ABTS is lowered to 0.7 mM, complete oxidation to the stable cation radical occurs in 30 min, thus circumventing the necessary overnight step. Using this format, it is now possible to accurately assess antioxidant activity based on the potassium persulfate/ABTS format in less than one hour which includes formation time of a stable ABTS radical. This methodology documented the presence of antioxidant properties of plant extracts used in Traditional Chinese Medicine. The degree of antioxidant activity was directly related to the extraction method. Greater antioxidant activity was associated with butanol extraction. When incorporated into a microtiter plate format, it supported rapid assessment of multiple determinations of dilutions of plant extracts in less than one hour which included time required for formation of a stable ABTS radical. The ease, improved time prerequisites, and minimal reagent needs with the microtiter plate format, makes this design attractive. It would prove of particular interest to individuals engaged in both smaller and high-volume throughput antioxidant assays of food and health products, and other biological fluids and tissues.

Beta-Glucuronidase (GusA) reporter enzyme assay for Escherichia coli

The beta-Glucuronidase (GusA) is a long-known reporter enzyme for many different species [1]. The E. coli gusA gene is often used in plant research because plants lack an endogenous gusA gene. In E. coli, the transcript of the gusA gene is more stable than that of the highly used reporter gene beta-galactosidase (lacZ) [2]. The GusA activity can be determined using the artificial substrate p-nitrophenyl-β-D-glucopyranosid (pNPG). pNPG is converted to glucoronic acid and para-nitrophenol (pNP), which can be quantified spectrometrically at 405 nm. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal gusA gene, which is available e.g. at the Keio collection [3]. The gusA gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the gusA gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

Dihydrofolate reductase (DHFR) reporter enzyme assay for Haloferax volcanii

The dihydrofolate reductase (DHFR) is routinely used a reporter enzyme for H. volcanii. The DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate and the concomitant oxidation of NADPH to NADP+. This leads to a reduction of extinction at 340 nm, which is measured to quantify the DHFR activity. To avoid background, it is best to use an H. volcanii strain with a deletion of the chromosomal dhfr gene, which is available upon request ([email protected]). However, the expression level of the chromosomal dhfr gene is very low, so that it is also possible to use the wildtype strain and subtract the DHFR background level. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the dhfr gene in a translational fusion with the gene of interest.

Glycerol-3-phosphate dehydrogenase (GlpD) reporter enzyme assay for Escherichia coli

Glycerol-3-phosphate dehydrogenase (GlpD) is a recently introduced reporter enzyme for E. coli [1]. GlpD calalyzes the oxidation of Glycerin-3-phosphate (G3P) to dihydroxyacetone-phosphate (DHAP). The oxidation is coupled to the reduction of the artificial yellow substrate tetrazol-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) to a blue-violet formazan, which is mediated by the electron carrier phenazin-methanosulfate (PMS). This leads to an increase in absorption at 570 nm, which is measured to quantify the GlpD activity. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal glpD gene, which is available e.g. at the Keio collection [2]. The glpD gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the glpD gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

A Novel Microtiter Plate Format High Power Open Source LED Array

Many photochemical or photobiological applications require the use of high power ultraviolet light sources, such as high-pressure mercury arc lamps. In addition, many photo-induced chemical, biochemical and biological applications require either a combinatorial setting or a parallel assay of multiple samples under the same environmental conditions to ensure reproducibility. To achieve this, alternative, controllable light sources, such as ultraviolet light emitting diodes (UV LEDs) with high power and spatial control are required. Preferably, LEDs are arranged in a suitable standardized 96-well microtiter plate format. We designed such an array and established the methods required for heat management and enabling stable, controllable illumination over time.

Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor

N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF’s divergent effects on FPR1 phosphorylation as well as PMN function.

In Vitro and in Vivo Antiplasmodial Activity of Extracts from Polyalthia suaveolens, Uvaria angolensis and Monodora tenuifolia (Annonaceae)

The present study aimed at investigating the in vitro and in vivo susceptibility of malaria parasites to crude extracts and fractions from Polyalthia suaveolens, Uvaria angolensis, and Monodora tenuifolia. The ethanolic extracts were prepared by maceration, and were further partitioned using water, dichloromethane, hexane, and methanol. The most promising fraction was subjected to column chromatography and the sub-fractions tested for activity in vitro. The antiplasmodial effect of extracts and fractions was tested against the Chloroquine resistant (PfK1) strain in 96 wells microtiter plate format using SYBR green florescence assay. The promising fraction was further assessed for cytotoxicity and acute toxicity in Swiss albino mice and subsequently against the rodent malaria parasite, P. berghei. Qualitative phytochemical screening was also performed on the promising fraction. The methanol fractions exerted the overall better effect with that of the twigs of P. suaveolens (PStw(Ace)) showing the highest potency with a IC50 value of 3.24 &micro;g/mL followed by the fractions of leaf of M. tenuifolia (MoTel(Ace), IC50= 3.84 &micro;g/ml) and stem bark of P. suaveolens (IC50= 4.90 &micro;g/ml). The phytochemical screening showed the presence of alkaloids, lactones, and phenols in the more active fraction of P. suaveolens (PStw(Ace)). The chromatographic fractionation of this fraction led to 12 sub-fractions with PS8 sub-fraction being the most active (IC50= 4.42 &micro;g/mL). In vivo, oral administration of 2000 mg/kg b.w of fraction PStw(Ace) in mice showed no signs of toxicity. Also, fraction PStw(Ace) at 400 mg/kg b.w exerted the highest suppressive effect against P. berghei strain B throughout the 4 days experiment (% parasitaemia below 5.2%). Overall, the results achieved supported the use of the three plants in the traditional treatment of malaria in Cameroon. More interestingly, the methanolic fraction of the twigs extract from P. suaveolens might be of interest in future development of an antimalarial phytodrug.