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N M Alexander

Publications and source records attributed to N M Alexander.

At least 19 recordsLinked to original sources
PubMed1990

Colorimetry and constant-potential coulometry determinations of transferrin-bound iron, total iron-binding capacity, and total iron in serum containing iron-dextran, with use of sodium dithionite and alumina columns.

After the parenteral administration of iron-dextran (imferon), the increased total iron concentrations in serum can be determined by atomic absorption spectroscopy and by colorimetric methods involving sodium dithionite, which reductively dissociates iron from the dextran complex. We report that constant-potential coulometry detects only about 55-70% of dextran-bound iron before dithionite reduction and variable amounts after reaction with the reducing agent. In addition, we have developed a procedure for determining transferrin-bound iron, total iron-binding capacity (TIBC), total iron, and dextran-bound iron with the Kodak Ektachem colorimetric system. In determining total serum iron, the sample is first mixed with sodium dithionite, which rapidly dissociates all dextran-bound iron, but does not remove iron from either transferrin or hemoglobin. After the mixture is applied to an Ektachem slide, transferrin-bound iron is released at pH 4 and is detected together with the iron previously bound to dextran. TIBC is determined by mixing serum with ferric citrate in moderate excess and filtering through a small alumina (Al2O3) column, which binds excess free iron and iron-dextran; the iron in the column eluate represents the TIBC. Transferrin-bound iron is determined by applying diluted serum without added ferric citrate to an alumina column and measuring the iron in the column eluate. Dextran-bound iron is equivalent to the difference between total and transferrin-bound iron. Using this method, we found that transferrin iron-binding sites are saturated in vitro by excess iron-dextran less efficiently than by ferric citrate.

Aluminum Oxide↗
PubMed1989

Inactivation of intracellular copper-zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation.

The copper chelator N,N'-diethyldithiocarbamate (DDC), is often used to inactivate intracellular copper-zinc superoxide dismutase in erythrocytes. However, in studies with red cells we found that the compound also reacted with oxyhemoglobin to produce oxygen radicals in addition to generating lipid peroxidation products, oxidized N,N'-diethyldithiocarbamate, methemoglobin, and sulfhemoglobin. Moreover, intracellular glutathione was depleted and vital cellular enzymes were susceptible to inactivation. We, and others, have confirmed these findings in nonerythrocytic cell lines. Thus, cells exposed to DDC are severely damaged before studies on the effects of added putative superoxide producing compounds can be performed with them. In this report, we have systematically investigated other copper chelators for their ability to inactivate intracellular copper-zinc superoxide dismutase without producing the deleterious effects mentioned above. Catechol, triethylenetetramine, and tetraethylenepentamine were found to be such agents when erythrocytes were dialyzed in the cold against dilute solutions of these chelators. In addition, with a myeloid leukemic cell line (HL-60), triethylenetetramine inhibited SOD without causing significant GSH oxidation. Examination of the affinity constants of chelators active against purified copper-zinc superoxide dismutase indicated that an affinity binding constant (log K1) between 12.6 and 13.8 was required for the chelator to successfully remove copper from the enzyme.

Cell Line↗
PubMed1988

Methylene blue competes with paraquat for reduction by flavo-enzymes resulting in decreased superoxide production in the presence of heme proteins.

Methylene blue competes 100 to 600 times more effectively than paraquat for reduction by three different flavo-containing enzymes; xanthine oxidase, NADH cytochrome c reductase, and NADPH cytochrome c reductase. Paraquat and methylene blue both interact with deflavo xanthine oxidase, indicating that neither electron acceptor reacted at the FAD site of the enzyme where molecular oxygen is reduced to superoxide. As the paraquat radical also directly reduced acetylated cytochrome c the hemeprotein could not be utilized for measuring superoxide production in the presence of the herbicide. In the presence of cytochrome c the methylene blue caused a sharp decrease in both paraquat-induced superoxide and hydroxyl radical production.

Cytochrome Reductases↗
PubMed1988

Potential of methylene blue to block oxygen radical generation in reperfusion injury.

Methylene blue interacts with xanthine oxidase at the iron-sulfide site in the electron pathway (Scheme I) that is known to serve as an electron-sink connecting the reductive and oxidative sites in both the oxidase and dehydrogenase forms. Thus, shunting of electrons to methylene blue at this site effectively diverts their flow away from the FAD site where molecular oxygen is converted to superoxide radicals. Since the electron affinity constants of xanthine oxidase for electron acceptors are FAD greater than iron/sulfide greater than molybdenum, methylene blue falls between the FAD and iron-sulfide site. Thus, methylene blue effectively inhibits superoxide and hydroxyl radical production while accelerating the conversion of xanthine to uric acid. As methylene blue is already approved for medicinal use in humans and is relatively nontoxic, the drug may have a role in reducing tissue injury associated with reperfusion. We are currently investigating this possibility in animal models.

Animals↗
PubMed1988

Definitive liquid-chromatographic demonstration that N-ethylglycine is the metabolite of lidocaine that interferes in the Kodak sarcosine oxidase-coupled method for creatinine.

Patients receiving lidocaine may show false increases of serum creatinine as assayed by the single-slide method on the Kodak Ektachem 700. Bissell et al. (Clin Chem 1987;33:951) suggested that this interference was due to oxidation of N-ethylglycine (NEG), a previously uncharacterized metabolite of lidocaine, by the sarcosine oxidase preparation used in the Ektachem creatinine slide. To investigate this possibility, we synthesized NEG, added it to drug-free human serum, and analyzed the NEG-supplemented sera for creatinine with the Ektachem 700. We found the following linear relationships between creatinine bias (y, mg/L) and NEG concentration (x, mg/L) for first (I), third (III), and fourth (IV) generation slides: I: y = 1.70x - 0.8 mg/L (n = 13, r = 1.0) III: y = 0.39x - 0.3 mg/L (n = 3, r = 1.0) IV: y = 0.79x - 1.8 mg/L (n = 13, r = 1.0) Using HPLC, we directly demonstrated the presence of NEG in sera of patients receiving lidocaine and quantified NEG concentrations in sera from four of these patients. The increasing artifactual bias in creatinine with increasing NEG concentration unequivocally confirmed that NEG is responsible for the lidocaine-associated interference in the Kodak Ektachem single-slide creatinine method.

Autoanalysis↗
PubMed1987

Carbamazepine (Tegretol) inhibits in vivo iodide uptake and hormone synthesis in rat thyroid glands.

Decreased serum concentrations of T3 and T4 occur in patients treated with the anticonvulsant drug carbamazepine (CBZ), but with rare exception, these patients remain euthyroid. The mechanism that accounts for diminished hormone levels is unknown, and our objective was to study the direct effect of CBZ on iodide uptake and hormone synthesis in thyroid glands of CBZ-treated and pair-fed control rats. Chronic ingestion (per os) of CBZ in male rats reduced the four hour thyroid 131I-iodide uptake by approximately 60%. This inhibition occurred after the animals had received sufficient CBZ to attain plasma CBZ concentrations of 0.8 microgram/ml. Continued treatment with CBZ ranging from 560 to 800 mg/kg/day for 14 days did not result in further inhibition of iodide uptake even though the plasma CBZ concentrations had increased 6-20 fold. No inhibition of iodide uptake was apparent when the animals initially received CBZ ranging from 40 to 152 mg/kg body weight for 22 days when there were no detectable levels of plasma CBZ. Overall growth rates of CBZ-treated rats were slightly (6-10%) less than the pair-fed control animals. Plasma T4 concentrations were reduced by 18% (p less than 0.05) in the CBZ-fed animals, while T3 concentrations were diminished by 53% (p less than 0.01). CBZ appeared to alter thyroidal iodide transport because the thyroid:plasma iodide ratios were decreased by 26% in the drug-treated rats. The distribution of radioiodine in thyroidal iodoamino acids was essentially the same in both groups of rats but the absolute quantities of radioiodine were more than 2.5 times greater in the control rats. CBZ failed to inhibit peroxidase-catalyzed iodide and guaiacol oxidation in vitro. No differences in thyroid gland morphology were noted in the two groups of animals, but weights of the glands from the CBZ-fed animals increased by 25% after 42 days. It is concluded that CBZ, or its metabolites, has a direct inhibitory effect on iodide utilization and hormone synthesis by the thyroid gland.

Animals↗
PubMed1987

Endocrine hormone abnormalities in Amanita poisoning.

Mushroom poisoning from the genus Amanita is being reported with increasing frequency in the United States. Endocrine hormone abnormalities were investigated in four patients who ingested mushrooms of the Amanita genus ("death cap", "destroying angel"). Marked abnormalities were found in the hormones controlling glucose, calcium, and thyroid homeostasis. Insulin and C-peptide concentrations were elevated at admission, indicating that the hypoglycemia associated with Amanita poisoning may not be solely secondary to hepatic failure. Serum calcitonin concentrations were elevated in conjunction with hypocalcemia. Parathyroid hormone concentrations (both the carboxyl- and amino-terminal assays) increased with time, but began returning to baseline as the hypocalcemia disappeared. The thyroxine concentrations were depressed in all four patients, and triiodothyronine (T3) concentrations were undetectable in three patients. Thyroid-stimulating hormone concentrations were never elevated, reflecting an unresponsive pituitary-hypothalamic axis to the development of hypothyroidism or a euthyroid-illness syndrome.

Adult↗
PubMed1986

Inhibition of erythrocyte superoxide dismutase by diethyldithiocarbamate also results in oxyhemoglobin-catalyzed glutathione depletion and methemoglobin production.

N,N-Diethyldithiocarbamate (DDC), a copper-chelating agent, not only inhibits superoxide dismutase activity in the red cell, but also depletes glutathione and promotes the production of methemoglobin, sulfhemoglobin, and small amounts of lipid peroxidation products. DDC reacts with oxyhemoglobin to yield disulfiram, hydrogen peroxide, and methemoglobin. Disulfiram and hydrogen peroxide both convert GSH to GSSG, while DDC reduces methemoglobin to oxyhemoglobin. Although disulfiram also reacts with the hemoglobin sulfhydryl groups, this reaction does not play a role in the conversion of GSH to GSSG. Other hemoglobin derivatives, ferrous, and ferric ions do not catalyze the oxidation of GSH by DDC. These results support the conclusion that DDC reacts with the super-oxo-ferriheme complex of oxyhemoglobin to generate hydrogen peroxide and disulfiram and that the cyclic conversion of oxyhemoglobin to methemoglobin and DDC and disulfiram results in the net oxidation of GSH. Thus, damage to DDC-treated erythrocytes exposed to a putative superoxide-generating toxin, such as 1,4-naphthoquinone-2-sulfonate, may actually be due to diminished GSH concentration and hemoglobin oxidation rather than to superoxide radicals. Glucose added to the incubation medium of DDC-treated erythrocytes fully prevented glutathione depletion but not the oxidation of oxyhemoglobin to methemoglobin. Several other copper-chelating agents either failed to inhibit the activity of purified superoxide dismutase or when incubated with erythrocytes produced more extensive GSH depletion and hemoglobin oxidation than DDC. It is concluded that the interpretation of results with erythrocytes exposed to copper-chelating agents must consider their effects on GSH and hemoglobin as well as on superoxide dismutase inhibition. Moreover, one must be mindful of the interference by DDC in the analysis of GSH with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of sufficient quantities of metaphosphoric acid to destroy DDC and that contamination of DDC with trace quantities of disulfiram may be a significant problem.

Copper↗
PubMed1985

Methylene blue directly oxidizes glutathione without the intermediate formation of hydrogen peroxide.

Methylene blue stimulates the oxidation of glutathione in red blood cells in vitro and in vivo. This oxidation has been attributed to hydrogen peroxide that is generated from the autooxidation of leucomethylene blue arising from the reduction of methylene blue by NADPH. In this report we present evidence that methylene blue directly oxidizes glutathione and that oxidation of glutathione by hydrogen peroxide is a secondary reaction. Moreover, superoxide dismutase has no effect on the oxidation. Under aerobic conditions, methylene blue oxidizes glutathione 30 times faster than the spontaneous autooxidation of glutathione. Under anaerobic conditions the stoichiometry of the reaction of methylene blue with glutathione supports a direct chemical reaction. The reaction rates between glutathione and methylene blue suggest a second order reaction over the conditions tested. That neither oxygen radical formation nor significant amounts of hydrogen peroxide are produced by methylene blue, even in the presence of added glucose, is further confirmed by the failure to detect significant amounts of lipid peroxidation products, or hemolysis, in red blood cells incubated with the dye.

Chemical Phenomena↗
PubMed1985

Rapid separation and identification of myoglobin and hemoglobin in urine by centrifugation through a microconcentrator membrane.

Urinary myoglobin (Mr = 16 500) is readily separated from hemoglobin (Mr = 64 000) by centrifugation through a microconcentrator membrane with a 30 000-Da cutoff. Myoglobin, but not hemoglobin, will pass through the membrane, after which each fraction may be separately analyzed by spectrophotometry. This technique is advantageous over direct spectrophotometry of urine because it is not restricted to analyzing the oxy forms of these hemoproteins and obviates the difficulty of discriminating small (3 nm) differences in their spectra. In addition, this method of separation is more complete than that attained by differential "salting out" with ammonium sulfate, and it is simpler than gel filtration or ultrafiltration under reduced pressure.

Centrifugation↗
PubMed1982

Fetal hemoglobin: optimum conditions for its estimation by alkali denaturation.

Alkali denaturation is the most commonly used technic to estimate fetal hemoglobin in red blood cells. The Betke and colleagues method (Nature 184: 1959; 1877-78) using cyanmethemoglobin (HiCN) was recommended by an International Committee for Standardization in Hematology for fetal hemoglobin levels between 2 and 40%. We showed that precision with samples containing up to 5% fetal hemoglobin can be considerably improved by measuring the absorbance of HiCN at 420 nm rather than at 540 nm because the molar absorptivity is 10 times greater in the Soret band. To determine optimum conditions for the assay, we studied the kinetics and stoichiometry of conversion of hemoglobin to HiCN with various concentrations and mixtures of ferricyanide, kinetics of alkali denaturation, ammonium sulfate precipitation of denatured HiCN, filtration as opposed to centrifugation for separating denatured HiCN, stability of HiCn in water and KCN solutions, and linearity of absorbance in the Soret band. With our modified procedure, the CV with normal amounts (less than 1%) of fetal hemoglobin improved from 28% to 8.7%. The CVs with 6.8% and 55% fetal hemoglobin were 7.0-4.4% respectively, and linear estimates were obtained with cells containing up to 50% fetal hemoglobin. We conclude that our modified alkali denaturation procedure yields reliable and reproducible estimates of fetal hemoglobin over a wide range of concentrations.

Adult↗
PubMed1981

Protein-linked iodotyrosines in serum after topical application of povidone-iodine (Betadine).

Markedly elevated serum PBI levels occur after therapy with povidone-iodine (Betadine), an iodine-polyvinylpyrrolidone iodophor. In this study, we investigated the serum iodine compounds from a severely burned patient with normal initial thyroid function tests who was swabbed with Betadine ointment and received daily therapeutic baths in Betadine. Three and 9 days after therapy, his serum contained 93 and 168 micrograms PGI/dl, respectively (normal range, 4-8), while the serum T4 and free T4 index were normal; the serum T3 level, however, was abnormally depressed. Most of the PBI was in albumin, and hydrolysis of the serum proteins with proteases released 35% of the PBI as monoiodotyrosine, 3.2% as diiodotyrosine, 0.01% as T3, and 2.5% as T4, as determined by competitive radioassays, anion exchange, and reversed phase high pressure liquid chromatography. The same concentrations of T4 and T3 were detected before and after hydrolysis. Failure of the proteases to completely hydrolyze iodoalbumin partially explains why all of the PBI was not recovered as iodotyrosines in the serum protein hydrolysates. Povidone-iodine rapidly iodinated tyrosine residues in human serum albumin at pH 7.4 and 37 C in vitro, and the ratio of diiodotyrosine to monoiodotyrosine increased as the molar ratio of povidone-iodine to albumin was increased. It is concluded that the abnormal increase in serum PBI resulted from absorption of the iodophor into the blood where it primarily iodinated albumin and, to a lesser extent, the globulins.

Administration, Topical↗