Eva F. Gudgin Dickson

TIME-RESOLVED DETECTION OF LANTHANIDE LUMINESCENCE FOR ULTRASENSITIVE BIOANALYTICAL ASSAYS

The principles and practice of the application of time-resolved lanthanide chelate luminescence (or fluorescence) as a detection method for ultrasensitive bioanalytical assays such as immunoassays and nucleic acid hybridization assays are reviewed. The various lanthanide chelate-based detection systems which have been developed for use in heterogeneous and homogeneous assay formats are described, including reagents, assay methods, and instrumentation, along with recent improvements in these methods. Detection systems described include those based on dissociative enhancement of lanthanide ions, direct labeling with luminescent chelates, enzyme-amplified lanthanide luminescence, lanthanide luminescence quenching, and energy transfer.

Ultrasensitive bioanalytical assays using time-resolved fluorescence detection

This article reviews the use of time-resolved fluorimetric detection of lanthanide chelate luminescence as a detection method for ultrasensitive bioanalytical assays. Assay formats and detection methods, and the principle of time-resolved fluorimetric detection, are described. Detection systems, assay formats, reagents, and instrumentation for time-resolved fluorimetric detection are outlined. A review of published and commercially available immunoassays and DNA hybridization assays using time-resolved fluorimetric detection of lanthanide chelate luminescence is given.

Formation of water-soluble porphyrins and protoporphyrin IX in 5-aminolevulinic-acid-incubated carcinoma cells

The bioconversion of 5-aminolevulinic acid (ALA) into hydrophobic protoporphyrin IX and other water-soluble porphyrins was investigated in Ehrlich ascite carcinoma (EAC) cells and in a myeloma cell line. The effects of irradiation (514 nm), temperature, incubation time and added glucose on the relative porphyrin concentrations (protoporphyrin vs. water-soluble porphyrins) were examined. Variations in these parameters induced a change in the amount of water-soluble porphyrins relative to protoporphyrin IX. The main component of the hydrophilic porphyrins was found to be uroporphyrin (Up), with minor components of coproporphyrin (Cp) and other carboxyporphyrins. The enhanced production of water-soluble porphyrins appears to be associated with alterations in the activities of the various enzymes in the heme biosynthetic pathway, resulting, for example, in the reduction in the activity of mitochondrial enzymes.

An affordable, portable fluorescence imaging device for skin lesion detection using a dual wavelength approach for image contrast enhancement and aminolaevulinic acid-induced protoporphyrin IX. Part II. In vivo testing.

Abstract. A fluorescence imaging prototype for skin lesion detection and diagnosis using aminolaevulinic acid (ALA) induced protoporphyrin IX (PpIX) was tested in vivo and in the clinic. The prototype was designed as an affordable, portable device to allow contrast enhanced imaging of skin lesions using either the dual excitation wavelength method or the dual emission wavelength method or both. In this study the prototype was tested first on an animal model. Topical application of ALA on defined spots on mouse skin gave PpIX fluorescence after about 3 hours of application. After successful in vivo testing the instrument was tested on basal cell carcinoma patients before ALA-PpIX photodynamic therapy. The patients were topically applied with ALA. After three hours the device was tested (immediately before treatment). The prototype showed good results in terms of contrast enhancement (elimination of unwanted background signals, e.g. autofluorescence) using either contrast enhancement method, both methods achieving similar results. The results achieved in this study, combined with the affordable design of the device, seem to allow cost-effective, contrast-enhanced imaging of skin lesions before or during photodynamic therapy using ALA induced PpIX.

An affordable, portable fluorescence imaging device for skin lesion detection using a dual wavelength approach for image contrast enhancement and aminolaevulinic acid-induced protoporphyrin IX. Part I. Design, spectral and spatial characteristics.

Abstract. Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancerous lesions. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals like autofluorescence and scattering can decrease the image quality and hence the diagnosis potential of this imaging method. A method involving two excitation and/or emission wavelengths was used in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. A fluorescence imaging device prototype was assembled using both the two wavelength excitation method and the two wavelength emission method. Additionally, a white light source was included to allow the collection of images as seen with the naked eye. The prototype was designed to be affordable and portable and was laid out towards the diagnosis of skin lesions using aminolaevulinic acid-induced protoporphyrin IX (PpIX). This paper describes the excitation and detection characteristics of a fluorescence imaging device prototype. This includes spectral and spatial characteristics of the various light sources included in the device as well as specifications of the image detector used. Furthermore, the image analysis procedure used for the dual wavelength excitation/emission is described.

Determination of the in vivo pharmacokinetics of palladium-bacteriopheophorbide (WST09) in EMT6 tumour-bearing Balb/c mice using graphite furnace atomic absorption spectroscopy.

Palladium-bacteriopheophorbide (WST09), a novel bacteriochlorophyll derivative, is currently being investigated for use as a photodynamic therapy (PDT) drug due to its strong absorption in the near-infrared region and its ability to efficiently generate singlet oxygen when irradiated. In this study, we determined the pharmacokinetics and tissue distribution of WST09 in female EMT6 tumour-bearing Balb/c mice in order to determine if selective accumulation of this drug occurs in tumour tissue. A total of 41 mice were administered WST09 by bolus injection into the tail vein at a dose level of 5.0 +/- 0.8 mg kg(-1). Three to six mice were sacrificed at each of 0.08, 0.25, 0.5, 1.0, 3.0, 6.0, 9.0, 12, 24, 48, 72, and 96 h post injection, and an additional three control mice were sacrificed without having been administered WST09. Terminal blood samples as well as liver, skin, muscle, kidney and tumour samples were obtained from each mouse and analyzed for palladium content (from WST09) using graphite furnace atomic absorption spectroscopy (GFAAS). The representative concentration of WST09 in the plasma and tissues was then calculated. Biphasic kinetics were observed in the plasma, kidney, and liver with clearance from each of these tissues being relatively rapid. Skin, muscle and tumour did not show any significant accumulation at all time points investigated. No selective drug accumulation was seen in the tumour and normal tissues, relative to plasma. Thus the results of this study indicate that vascular targeting resulting from WST09 in the circulation, as opposed to selective WST09 accumulation in tumour tissues, may be responsible for PDT effects in tumours that have been observed in other WST09 studies.

ON THE SOURCE OF THE OSCILLATIONS OBSERVED DURING in vivo ZINC PHTHALOCYANINE FLUORESCENCE PHARMACOKINETIC MEASUREMENTS IN MICE

Surface‐detected fluorescence spectroscopy can be used to monitor the pharmacokinetics of uptake and clearance of red‐absorbing fluorophores such as zinc(II) phthalocyanine (ZnPc) in vivo. When this technique is applied to mice that have been fed on a normal chlorophyll‐based diet, and particularly when measurements are performed in the abdominal region, oscillations are sometimes observed superimposed on the pharmacokinetic curve of the ZnPc. An oscillatory signal has also been observed arising from the abdominal region of control mice fed a normal diet but not injected with the ZnPc photosensitizer; this oscillatory component to the signal is reduced when mice are fed a chlorophyll‐free diet. The oscillatory signal component has been attributed to fluorescence arising from chlorophyll derivatives (pheophorbide/pheophytin) contained in the rodent food, whose concentration in the measured abdominal region changes substantially with time, presumably due to digestive processes. Thus it is important to be aware of the possibility of such artifactual contributions to in vivo fluorescence pharmacokinetic measurements.

In Vitro Dermal Absorption of Methyl Salicylate, Ethyl Parathion, and Malathion: First Responder Safety

In vitro tests with fresh dermatomed (0.3 to 0.4 mm thick) female breast skin and one leg skin specimen were conducted in Bronaugh flow-through Teflon diffusion cells with three chemicals used to simulate chemical warfare agents: 14C-radiolabeled methyl salicylate (MES), ethyl parathion (PT), and malathion (MT), at three dose levels (2, 20, and 200 mM). Tests were conducted at a skin temperature of 29°C using a brief 30-min exposure to the chemical and a 6.5-h receivor collection period. Rapid absorption of all three chemicals was observed, with MES absorbed about 10-fold faster than PT and MT. For MES, PT, and MT, respectively, there was 32%, 7%, and 12% absorption into the receivor solution (Hanks HEPES buffered saline with 4% bovine serum albumin [BSA], pH 7.4) at the low dose (2 mM), 17%, 2%, and 3% at the medium dose (20 mM), and 11%, 1%, and 1% at the high dose (200 mM) levels. Including the skin depot for MES, PT, and MT, respectively, there was 40%, 41%, and 21% (low dose), 26%, 16%, and 8% (medium dose), and 13%, 19%, and 10% (high does) absorption. Efficacy of skin soap washing conducted at the 30 min exposure time ranged from 31% to 86%, varying by chemical and dose level. Skin depot levels were highest for the relatively lipophilic PT. “Pseudo” skin permeability coefficient (K p) data declined with dose level, suggesting skin saturation had occurred. An in-depth comparison with literature data was conducted and risk assessment of first responder exposure was briefly considered.

A new whole-body vapor exposure chamber for protection performance research on chemical protective ensembles

A chemical vapor exposure chamber was designed to permit the study of whole-body vapor exposure of individuals wearing full protective clothing and equipment systems. A methodology also was developed to quantify the vapor protection performance of chemical protective ensembles (CPE) under safe and validated laboratory procedures. The principal research objectives were to (1) provide a methodology to accurately assess the performance of CPE and equipment under different environmental and chemical vapor challenge conditions; (2) quantify the vapor protection on a per body region basis; (3) have a systems level tool to aid in the research and development of more effective CPE for use in chemical biological environments; and (4) have a safe and reliable means of qualifying new CPE on the basis of vapor protection. Although designed for the evaluation of military-style protective equipment, the procedures apply equally to other styles of CPE used by civilian agencies such as firefighters, police, and hazmat units. The chamber and methodology were specifically designed to examine the vapor protection performance of clothing ensembles, including the details of protection variation over the body. A variety of exposure conditions appropriate to indoor and outdoor scenarios are possible, including the effects of wind, temperature, and relative humidity. Protection performance results from a number of individuals wearing typical military-style CPE are presented. These results demonstrate that there is no such thing as a unique protection performance level obtained for a given CPE. Rather, the individual and the ensemble interact differently in each situation, resulting in a protection performance distribution for individuals, and for groups of wearers, even under a standardized set of exposure conditions.

Estimates of Percutaneous Toxicity of Sulfur Mustard Vapor Suitable for Use in Protective Equipment Standards

An analysis was performed of historical human chamber data for exposure to sulfur mustard vapor, in order to correlate skin exposure dosages with effects in a manner specifically suitable for use in protective clothing standards. Data were reanalyzed to take into account (1) body region variability of skin responses to a single acute exposure to sulfur mustard vapor, (2) effect of hot/humid versus cooler exposure, and (3) influence of clothing. This approach permits deriving predicted skin responses pertinent to a protective clothing wearer, for a relatively short single acute exposure to vapor (up to a few hours) under the hot/humid conditions expected within a protective ensemble. Values for permissible dermal exposure to sulfur mustard vapor are proposed for protected emergency responders or military serving in combat theaters that may be used in standards intended to be employed in conjunction with evaluation of vapor protection provided by individual protective equipment for protection against chemical warfare agents by Man-in-Simulant vapor test methods.