Robert Wei

Reduction of iodonitrotetrazolium violet by superoxide radicals

p-Iodonitrotetrazolium (2-(4-iodophenyl-3-(4-nitrophenyl)- 5-phenyltetrazolium; INT) was reduced to a water-soluble product with an absorbance maxima at about 505 nm (reddish pink) by superoxide anion (O2-.) generated by xanthine/xanthine oxidase. The rate of INT reduction was linearly related to the xanthine oxidase activities, and was inhibited by superoxide dismutase. The soluble product may further be converted to an insoluble product, presumably nonionic formazan, with an absorbance maxima of 490 nm (purplish), under certain conditions, and the rate of the formazan formation depended on pH and protein concentration.

Measurement of total HDL, HDL2 and HDL3 by dextran sulfate—MgCl2 precipitation technique in human serum

We describe a simple and reliable method for determination of total HDL, HDL2, and HDL3 by a precipitation technique using dextran-sulfate (Mr 50,000)-Mg2+. A combined solution of dextran sulfate and Mg2+ at their respective final concentration of 0.9 g/l and 27 mmol/l was optimal for separating total HDL from the other lipoproteins. The present method compares favorably with a heparin-MnCl method (r = 0.998). The HDL was further resolved into HDL2 and HDL3 by addition of a combined solution of dextran sulfate and Mg2+ (1.5 m/l and 10 g/l) to the total HDL solution. Comparison of this precipitation method with the well-established ultracentrifugation method yielded the mean correlation coefficient of 0.941 and 0.869 for HDL2 and HDL3, respectively.

Maternal and neonatal urinary excretion of sulfate and glucuronide ritodrine conjugates.

Ritodrine is a β2‐adrenergic agonist that is used clinically for the management of preterm labor. The β2 activity of ritodrine produces the relaxation of smooth muscles and is believed to act directly on the β2‐receptors of the myometrium. Reports in the literature suggest that ritodrine is inactivated by sulfate and glucuronide conjugation, but this has not been verified in humans. Studies on animal models indicate that the sulfate conjugate is a major urinary metabolite of ritodrine. Recent investigations of maternal and neonatal urinary excretion of ritodrine indicate that 80% to 90% of the drug is in the form of conjugates. The purpose of this study was to determine the nature of these conjugates. Our study indicates that both the mother and neonate excrete glucuronide and sulfate conjugates of ritodrine. The sulfate conjugate accounts for 45% of maternal excretion and 66% of neonatal excretion; the glucuronide conjugate accounts for 38% and 23% of maternal and neonatal excretion, respectively. Significantly different metabolic profiles suggest that the neonate may be capable of forming conjugated metabolites of ritodrine.

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection.

Abstract We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.

Alcohol dehydrogenase-catalyzed reduction of protein carbonyl derivatives

A simple method for measuring protein carbonyl derivatives is described. The assay is based on the catalytic reduction of protein carbonyl derivatives by NADH-dependent alcohol dehydrogenase at pH 7.5. Bovine serum albumin, oxidized by a system that generates highly reactive hydroxyl radicals via a Fenton-type reaction, is reacted with NADH in the presence of the enzyme. The carbonyl derivatives are quantitated on the basis of absorbance changes at 340 nm using millimolar absorptivity of 6.2 mM-1 cm-1. The method correlates well with a method that utilizes 2,4-dinitrophenylhydrazine.

Differential expression of proteins induced by lead in the Dwarf Sunflower Helianthus annuus.

The Dwarf Sunflower (Helianthus annuus) is a hyperaccumulator of toxic metals including cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb). In order to identify stress response to Pb, plants were exposed to a mixture of 30 mg/l of three ions, Cd, Cr, and Ni, with and without Pb. Soluble proteins were resolved by two-dimensional gel electrophoresis. Four proteins were differentially expressed and very abundant in the leaf samples after plants were exposed to all these four metals. The first protein spot contained two proteins: chitinase and a chloroplast drought-induced stress protein CDSP-34. The second spot contained a thaumatin-like protein. Two proteins in spot 3 were identified as heat-shock cognate 70-1 and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Several peptides were identified in spot 4 but none could be matched to any sequence in the NCBI database.

A Simple Electrochemical Method for the Quantitative Determination of Acetaminophen in Serum

Abstract A simple electrochemical method for the determination of acetaminophen in serum is described. The eleotrode and associated electronics are simple, reliable, and inexpensive to build. The apparatus can be operated at a rate of 2–3 determinations per minute using only 10 μl serum per determination. The procedure includes extraction of acetaminophen in ethyl acetate and subsequent oxidative amperometric detection of the drug by a form of flow-injection analysis. The system parameters of buffer, pH, and redox potential have been optimized to permit measurement of less than 10 μg/ml of acetaminophen. The determination is linear over the range of 10–300 μg/ml with a C.V. of less than 3% for replicate analysis of the same sample.

Properties of creatine kinase-BB from canine and human brain tissues

Creatine kinase (EC 2.7.3.2) BB isoenzyme (CK-BB) was purified to homogeneity from canine and human brain tissues. The purified protein from both sources exhibits Mr of 84,700 daltons. The canine isoenzyme exhibits several properties similar to human isoenzyme with respect to reactive and total thiol groups, UV spectra, isoelectric points and reaction kinetics. While both canine and human CK-BB isoenzymes are unstable compared to other CK isoenzymes, canine CK-BB is even less stable than the human enzyme, losing most of its activity within 20 h at 4 degrees C at pH 5.0. Addition of 2-mercaptoethanol does not prevent rapid loss of the enzyme activity. Increasing the pH to 9.0, however, increases the stability of both CK-BB isoenzymes. Agarose electrophoresis demonstrated the presence of MM as well as BB isoenzyme in various parts of brain tissues. BB was present at an activity of 90.8-93.3 U/mg and MM at 6.7-9.2 U/mg.

Measurement of Superoxide Dismutase in Erythrocytes and Whole Blood Using Iodonitrotetrazolium Violet

Abstract A simple analytical method for superoxide dismutase (SOD) is described. It is based on the competitive reduction of a superoxide anion (O2 −) by piodonitrotetrazolium (2-[4-iodopheny]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride, INT). The magnitude of inhibition of INT reduction by SOD was dependent on the rate of O2 − generation by xanthine oxidase: within the range of xanthine oxidase we have examined (2.2 – 4.4 μg/mL or 1.1 – 2.2 mU), the inhibition ranged from 49 to 64% and was inversely related to the xanthine oxidase activity. With 4.4 μg/mL of xanthine oxidase, as high as 1.0 units (0.32 μg/mL) of SOD inhibited about 50% of INT reduction, and the calibration curve derived from the INT assay correlates quite well with that of the cytochrome c method (r = 0.991). Analysis of the patient samples (n = 19) for erythrocytes SOD by both the INT and the cytochrome c methods compares reasonably well (r = 0.901). Students t-test on the results shows no significant difference between the values o...

Purification of creatine kinase-BB isoenzymes from human and canine tissues☆

We describe a procedure for the purification of creatine kinase BB isoenzyme from canine and human brain tissue. The purification involves sequentially 50-70% (NH4)2SO4 fractionation, DEAE-Sephacel, Blue-Sepharose CL-6B, and chromatofocusing chromatography. This method produces a homogeneous protein purification as verified by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing chromatography.