Performance validation for microplate fluorimeters.

As the sophistication of instruments that make fluorimetric measurements on samples in microplates has increased, so has the need for methods to validate instrumental performance. This paper describes a solid-state validation microplate that tests multiple aspects of fluorescence performance, including signal linearity, gain, noise, sensitivity, wavelength accuracy, and polarization stability. Both the operating principles and the validation of the validation microplate are discussed.

Regeneration of functional hemoglobin from iron(III) hemoglobin by reduction with hydrogen and a heterogeneous catalyst.

Functional hemoglobin was regenerated from partially autoxidized hemoglobin by reduction with molecular hydrogen in the presence of a heterogeneous catalyst consisting of elemental platinum embedded in an electroactive polymer. The visible spectrum of the regenerated hemoglobin was identical to that of native iron(II) hemoglobin. The regenerated hemoglobin displayed highly cooperative oxygen-binding characteristics.

Substituent effects on the binding constants of arsenical-dithiol adducts.

Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to determine the relative binding constants for several arsenical-dithiol adducts. The compounds investigated were 2,3-dimercaptopropanol (British anti-lewisite; BAL), 1,2-ethane dithiol (ET), and 1,2-propane dithiol (PDT). It was found that PDT has a significantly higher affinity than ET or BAL for phenyldichloroarsine (PDA) in methanol.

Activation energies and formation rate constants for organic arsenical-antidote adducts as determined by dynamic NMR spectroscopy.

Phenyldichloroarsine reacts with 1,3-dimercapto-2-propanol and 1,2-dimercaptopropane to form 1:1 adducts in the form of a six-membered and five-membered heteroatom rings. Two geometric isomers for each compound are present in dynamic equilibrium. Rate constants and the activation barriers for the interconversion of the geometric isomers were determined by dynamic NMR spectroscopy.

Reduction of extracellular methemoglobin by erythrocytes.

Erythrocytes, suspended in a glucose-containing buffer, catalyzed the partial reduction of extracellular methemoglobin. Physiological concentrations of ascorbic acid or dehydroascorbic acid greatly enhanced the rate of reaction and the ultimate extent of reduction. The relationship between erythrocyte concentration and initial reaction rate was nonlinear, which suggested that the rate limiting factor was not an erythrocyte membrane enzyme.

Relative binding constants of arsenical-antidote adducts determined by NMR spectroscopy.

Proton nuclear magnetic resonance spectroscopy was used to determine relative binding constants for several arsenical-antidote adducts. It was found that BAL (2,3-dimercaptopropanol) and DMPS (2,3-dimercaptopropanesulfonic acid) had a higher affinity than DMSA (2,3-dimercaptosuccinic acid) for the two organic arsenicals studied.

2,3-Dithioerythritol, a possible new arsenic antidote.

British antilewisite (2,3-dimercaptopropanol; BAL) has long been used as an arsenic antidote, but its therapeutic efficacy is limited by its inherent toxicity. We synthesized two less toxic derivatives of BAL and investigated their potential as antidotes to organic arsenic. The new compounds, 2,3-dithioerythritol (DTE) and 2,2-dimethyl-4-(hydroxymethyl)-1,3-dithiolane (isopropylidene derivative of BAL), react readily with phenyldichloroarsine (PDA) to yield the expected corresponding cyclic 1,3-dithioarsolanes.

Reaction of trans-2-chlorovinylarsine oxide with polydeoxynucleotides.

Trans-2-chlorovinylarsine oxide (in DCl/acetone-d6) was added to various polydeoxynucleotides. The arsenical did react with poly[dG].poly[dC], releasing guanine, and resulting in a partial apurinic duplex.

Structure and dynamics of a lipoic acid-arsenical adduct.

The lipoic acid-phenyldichloroarsine adduct was prepared in methanol, and the structure and molecular motions of this adduct were studied. The results showed that a six-membered heteroatom adduct was formed. One-dimensional and two-dimensional NMR spectroscopy was used to confirm the structure and assign some of the resonances in the proton and carbon spectra.

The interaction of phenyldichloroarsine with erythrocytes.

The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.

Gas chromatographic analysis of urinary dimercaptosuccinic acid.

The therapeutic use of disulfhydryl compounds such as 2,3-dimercaptosuccinic acid (DMSA) for the treatment of heavy metal poisoning has generated a requirement for specific and sensitive methods to determine those compounds in biological media. We have developed a gas chromatographic assay for DMSA in urine. The use of capillary column technology eliminates the requirement for a preliminary clean-up step.

One-dimensional and two-dimensional nuclear magnetic resonance studies of the reaction of phenyldichloroarsine with glutathione.

14C-labeled phenyldichloroarsine (PDA) enters the red blood cell and forms a 1:2 adduct with intracellular glutathione. Upon gel filtration of the hemolysate, [14C]PDA was recovered with the glutathione-containing fractions. One-dimensional and two-dimensional nuclear magnetic resonance spectroscopy were used to confirm the structure of the adduct and elucidate its stereochemistry, stability, and reactivity.

Wholesomeness of Irradiated Foods.

The history and applications of food irradiation are reviewed. The term wholesomeness when applied to food irradiation, embodies the concepts of microbiological and toxicological safety, and nutritional adequacy. The status of these areas of concern is reviewed.

Histologic changes in nude mouse skin and human skin xenografts following exposure to sulfhydryl reagents: arsenicals.

This report documents the histological changes in nude mouse skin and in human skin xenografts on nude mice following exposure to phenyldichloroarsine (PDA), a vesicant arsenical. Under light microscopy, we observed in PDA-treated human skin grafts: 1) degeneration of epidermal cell nuclei (apparent by 2 hr after exposure with increasing severity through 48 hr); 2) loss of epidermal cytoplasmic basophilia (apparent by 4 hr, maximal within 12 hr); 3) epidermal cytoplasmic vacuolization (vacuoles appeared within 4 hr and increased in size through 24 hr); 4) cleft formation within the basement membrane zone (apparent by 12 hr, increasing in severity through 24 hr); 5) inflammation evidenced by polymorphonuclear leukocyte (PMN) infiltration (apparent by 4 hr and increasing through 48 hr). The PMNs frequently formed a wall around the lesion, but did not infiltrate the treated area.

A continuous-flow (AutoAnalyzer II) procedure for measuring erythrocyte transketolase activity.

In this semiautomated method, an AutoAnalyzer II is used to measure the enzymic production of glyceraldehyde 3-phosphate in hemolysates, to assay erythrocyte transketolase (EC 2.2.1.

Interactions between folate and ascorbic acid in the guinea pig.

Possible interactions between folic acid (folate) and ascorbic acid (AA) have been suspected because megaloblastic anemia is occasionally observed in scorbutic patients, and it may or may not respond to folate treatment. Male weanling guinea pigs were fed diets containing high levels of folate and AA or diets deficient in one or both vitamins. A total of 36 animals, including 9 controls, were studied.

Erythrocyte filterability, fragility, and membrane proteins in folic acid deficient guinea pigs.

The purpose of this study was to determine if erythrocytes from folic acid-deficient guinea pigs could be used as a model for the study of defects which have been reported in megaloblastic anemic patients. Reported defects include decreased deformability and abnormal membrane proteins. Red cell filterability was studied with polycarbonate sieves and cellulose filters.

The relationship of the urinary ascorbate metabolites to specific levels of ascorbate supplementation in the monkey.

This study was designed to investigate urinary ascorbic acid (AA) and its metabolites derived from (1-14C) AA administered to trained monkeys fed different levels of ascorbate for extended periods of time. A chromatographic procedure was developed which rapidly separates the urinary compounds into four major fractions with minimal degradation. The distribution of 14C in the four peaks was dependent upon the ascorbate nutritional status of the monkey and remained constant for at least 30 days postlabel.

Thiamin, riboflavin, and vitamin B6 contents of selected foods as served.

Information of thiamin, riboflavin, and vitamin B6 contents of eighty-one food items is presented. The data represent over two hundred samples collected from serving lines in dining halls of two military installations. Ninety per cent of the vitamin B6 values and 35 per cent of the thiamin and riboflavin diata are not available in the current literature.