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. P50 values for oxidized-regenerated hemoglobin samples were not different from native hemoglobin. The Hill coefficients for regenerated hemoglobin were slightly lower than the controls, possibly because of small amounts of irreversibly oxidized hemoglobin arising during the initial autoxidation. The advantages of the reduction system include: (1) the heterogeneous catalyst avoids the problem of protein adsorption onto bare platinum, (2) catalyst and reducing agent are easily removed from the protein, and (3) the by-product H+ is buffered easily.

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. The activation barriers indicate that the five-membered heteroatom ring is more stable than the six-membered heteroatom ring.

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. Also, significant dehydroascorbate-stimulated reduction occurred even when the erythrocytes and methemoglobin were separated by a dialysis membrane. The above observations indicate that the transfer of reducing equivalents across the erythrocyte membrane and reduction of extracellular methemoglobin can be accomplished by release and recycling of ascorbic acid.

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. The BAL derivatives were compared to BAL in terms of their cytotoxicity and their ability to rescue PDA-poisoned mouse lymphoma cells in culture. The dithiolane was not a good antidote in the cultured cell system. In contrast, DTE was less toxic than BAL or DMSA and was superior at improving cell survival in PDA-exposed cells.

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. Spin-lattice relaxation times of the various carbon atoms indicated that the overall molecular reorientation time (tau R) of the molecule is 0.02 ns at 30 degrees C. An Arrhenius plot of the data showed that the activation energy (Ea) for molecular tumbling is 13.4 kJ/mol.

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. Samples are first reduced electrochemically to liberate DMSA present as disulfides. The reduced product is then extracted into ethyl acetate and the organic phase removed by evaporation. The residue is derivatized with N,O-bis (trimethylsilyl) acetamide for gas chromatography. The silylated DMSA derivative is then detected with a flame ionization detector. The detection limit for DMSA is 1.9 nmol per 1-microliter aliquot of derivatized extract injected on column (detector sensitivity at 1.10(-11) A/mV). The utility of the method was demonstrated by analyzing the urine of rats orally dosed with DMSA.

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.

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. Nude mouse skin reacted faster to PDA than did the grafts, but the histological changes were similar. Nude mouse hair follicles and sebaceous glands showed similar cellular changes at approximately the same time as did epidermal cells. Transmission electron microscopy of mouse skin exposed to PDA revealed a widening of intercellular spaces with attenuation of desmosomes. The subepidermal clefts resulted from separation within the lamina lucida with the lamina densa forming the base of the cleft. Diphenylchloroarsine caused lesions histologically indistinguishable from those of PDA. Lesions resulting from exposure to other sulfhydryl-binding compounds were very different from arsenical lesions. The arsenical-sensitive cellular constituents were not identified.

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. Nutritional studies have addressed the effects of irradiation on nutrient content and bioavailability, and evaluation of potential consequences of changes in either. Results of rat studies are presented in which we tested for the presence of anti-thiamin and anti-pyridoxine activity in radappertized chicken and beef. Test meats were analyzed for thiamin and pyridoxine to establish a basis for incorporation into repletion diets. Thiamin levels in gamma- and electron-irradiated, and thermally processed (commercial canning) chicken were 74, 34 and 78%, respectively, of the vitamin level in a frozen meat reference; the levels in beef were 77, 56 and 79%, respectively. Pyridoxine levels in chicken were 50, 38 and 17%, respectively, of the reference level. Rats were depleted in each vitamin, then repleted at two vitamin levels with diets containing test meats. Activities of transketolase, aspartate aminotransferase and alanine aminotransferase in erythrocytes from these rats provided no consistent evidence of antivitamin presence. It was concluded that these irradiated meats pose no problem regarding vitamins B1 and B6 if part of a complete diet.

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 2.2.1.1) activity. Hemolysate and indicator reactions are separated by dialysis to eliminate hemoglobin interference and increase sensitivity. Internal standards of glyceraldehyde 3-phosphate in hemolysate carriers were quantitatively measured with good precision and accuracy in the presence or absence of the transketolase substrate, ribose 5-phosphate. Chart-recorder values for these standards were used to calibrate the AutoAnalyzer output in IUB units (U) of transketolase activity. Substrate-product relationships were examined to characterize reaction kinetics and optimize assay conditions. AutoAnalyzer transketolase results correlated well with those from two manual procedures.

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. When anorexia began to appear in the deficient groups, all animals were killed by exsanguination, and tissue samples (blood, liver, adrenal, kidney, spleen, and intestinal mucosa) were removed for AA and folate analyses. Folate and AA deficiency lowered tissue folate and AA levels, respectively. AA deficiency, either alone or in combination with folate restriction, did not affect tissue folate levels, nor did AA deficiency significantly exacerbate the anemia and leukopenia caused by folate deficiency. However, there was an unexpected decrease in AA levels in the liver and adrenal glands with folate deficiency. Although AA does not appear to be needed for normal folate metabolism, the lower AA levels associated with a folate deficiency are indicative of an interaction between the two vitamins.