Wayne Chris Hawkes

An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components.

A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4 degrees C prior to chromatography on a 5-microns octadecylsilyl column. AA concentrations (mean +/- SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 +/- 0.05, 15.2 +/- 6.28, and 2.43 +/- 1.63 micrograms/10(8) cells, respectively; the mean plasma AA concentration was 0.97 +/- 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 X SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.

Regulation of Redox Signaling by Selenoproteins

The unique chemistry of oxygen has been both a resource and threat for life on Earth for at least the last 2.4 billion years. Reduction of oxygen to water allows extraction of more metabolic energy from organic fuels than is possible through anaerobic glycolysis. On the other hand, partially reduced oxygen can react indiscriminately with biomolecules to cause genetic damage, disease, and even death. Organisms in all three superkingdoms of life have developed elaborate mechanisms to protect against such oxidative damage and to exploit reactive oxygen species as sensors and signals in myriad processes. The sulfur amino acids, cysteine and methionine, are the main targets of reactive oxygen species in proteins. Oxidative modifications to cysteine and methionine can have profound effects on a protein’s activity, structure, stability, and subcellular localization. Non-reversible oxidative modifications (oxidative damage) may contribute to molecular, cellular, and organismal aging and serve as signals for repair, removal, or programmed cell death. Reversible oxidation events can function as transient signals of physiological status, extracellular environment, nutrient availability, metabolic state, cell cycle phase, immune function, or sensory stimuli. Because of its chemical similarity to sulfur and stronger nucleophilicity and acidity, selenium is an extremely efficient catalyst of reactions between sulfur and oxygen. Most of the biological activity of selenium is due to selenoproteins containing selenocysteine, the 21st genetically encoded protein amino acid. The most abundant selenoproteins in mammals are the glutathione peroxidases (five to six genes) that reduce hydrogen peroxide and lipid hydroperoxides at the expense of glutathione and serve to limit the strength and duration of reactive oxygen signals. Thioredoxin reductases (three genes) use nicotinamide adenine dinucleotide phosphate to reduce oxidized thioredoxin and its homologs, which regulate a plethora of redox signaling events. Methionine sulfoxide reductase B1 reduces methionine sulfoxide back to methionine using thioredoxin as a reductant. Several selenoproteins in the endoplasmic reticulum are involved in the regulation of protein disulfide formation and unfolded protein response signaling, although their precise biological activities have not been determined. The most widely distributed selenoprotein family in Nature is represented by the highly conserved thioredoxin-like selenoprotein W and its homologs that have not yet been assigned specific biological functions. Recent evidence suggests selenoprotein W and the six other small thioredoxin-like mammalian selenoproteins may serve to transduce hydrogen peroxide signals into regulatory disulfide bonds in specific target proteins.

The effects of dietary selenium on the immune system in healthy men

Eleven men were fed foods naturally high or low in selenium for 120 d. Selenium intake was stabilized at 47 µg/d for 21 d, then changed to either 13 or 297 µg/d for 99 d, leading to significantly different blood selenium and glutathione peroxidase concentrations. Serum immunoglobulins, complement components, and primary antibody responses to influenza vaccine were unchanged. Antibody titers against diphtheria vaccine were 2.5-fold greater after reinoculation in the high selenium group. White blood cell counts decreased in the high-selenium group and increased in the low-selenium group, resulting primarily from changes in granulocytes. Apparent increases in cytotoxic T-lymphocytes and activated T-cells in the high-selenium group only approached statistical significance. Lymphocyte counts increased on d 45 in the high-selenium group. In vitro proliferation of peripheral lymphocytes in autologous serum in response to pokeweed mitogen was stimulated in the high-selenium group by d 45 and remained elevated throughout the study, whereas proliferation in the low selenium group did not increase until d 100. This study indicates that the immune-enhancing properties of selenium in humans are the result, at least in part, of improved activation and proliferation of B-lymphocytes and perhaps enhanced T-cell function.

Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteine, the most highly purified 75Se-fractions were greater than 100-fold purified relative to 35S. These fractions contained less than 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.

Measurement of Vitamin C in Blood Components by High-Performance Liquid Chromatography.: Implication in Assessing Vitamin C Status

The fact that platelets, PMN leukocytes, and MN leukocytes concentrate ascorbic acid suggests that vitamin C has an important role in their physiological functions. The question still remains as to which one of the cells best reflects vitamin C status. The ascorbic acid content of PMNs and platelets correlates positively with plasma concentration and supplementation with vitamin C, as shown in Evans et al. They also found that MN leukocytes, in contrast, do not show any such relationship; however, MN leukocytes maintain the highest levels of ascorbic acid and play a very important function in immunocompetence. We have found that with a limited number of subjects, ascorbic acid content of MN and PMN leukocytes correlates positively with plasma ascorbic acid, but there was no correlation between platelets and plasma ascorbic acid (unpublished results). Therefore, further work is necessary to evaluate these three blood components for the best cellular marker of vitamin C status. We have developed a reversed-phase HPLC method for ascorbic acid that can be used in conjunction with our cellular differential centrifugation technique for the determination of ascorbic acid in relatively pure blood cell fractions. The chromatographic method is simple, sensitive, and automated. It clearly resolves ascorbic acid, which is the major form of the vitamin found in vivo and is not prone to interference by sugars, carbohydrates, or nucleotides.

Plasma selenium decrease during pregnancy is associated with glucose intolerance

There is an increased requirement for selenium during pregnancy, presumably for fetal growth, which manifests as decreasing maternal blood and tissue selenium concentrations. These decreases are greater in pregnant women with gestational or preexisting diabetes. We measured selenium status and glucose tolerance between wk 12 and 34 of gestation in 22 pregnant women. We found that the increase in blood glucose in response to an oral glucose challenge at 12 wk gestation and the increase in fasting glucose during pregnancy were inversely correlated with plasma selenium concentration. Women with lower plasma glutathione peroxidase activities during pregnancy also tended to have higher fasting glucose levels. These inverse relationships between selenium status and glucose tolerance are consistent with earlier observations that suggest a link between selenium and glucose metabolism. The observation that changes in serum glucose were not accompanied by changes in insulin suggests that selenium may affect glucose metabolism downstream from insulin, or through independent energy regulatory pathways such as thyroid hormone.

Selenocysteine-containing proteins and glutathione peroxidase

Publisher Summary This chapter present the methods currently used in laboratory to study glutathione peroxidase and other selenoproteins of the rat tissues. The principal techniques described are the in vivo labeling of selenoproteins with 75 Se and the chromatographic fractionation of either native or denatured selenoproteins from various tissues for identification by elution pattern and apparent molecular weight. The chapter also describes the use of tryptic digests of selenoproteins to obtain selenopeptides that allow further definition of various selenoproteins. Although all the selenocysteine-containing proteins in the rat presumably have an important biological function, the only mammalian selenoprotein that has an assay for its activity is glutathione peroxidase. At the current state of knowledge, the only way to assay other selenoproteins is to measure the selenium content of separated protein fractions. Because it is very convenient to follow selenium with 75 Se, various methods were developed to allow the simultaneous assay of several 75 Se-labeled selenoproteins from most types of mammalian tissues. The technique of covalent chromatography on Sepharose has been used to purify glutathione peroxidase. Selenocysteine serves as the ligand that binds glutathione peroxidase.

In vitro synthesis of glutathione peroxidase from selenite Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase.

Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway

Background: Selenium may prevent or help cause cancers, but the mechanisms are unclear. Results: Silencing MKK4, JNK2, p38γ, or p38δ rescued G1 arrest from SEPW1 depletion. Conclusion: G1 arrest from SEPW1 silencing is mediated by a MKK4-p38γ/p38δ/JNK2-p53 signaling pathway. Significance: The function of SEPW1 in cell cycle regulation is important to understanding the role of selenium in carcinogenesis. Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G1 arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G1 arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G1 arrest.

Automated continuous-flow colorimetric determination of glutathione peroxidase with dichloroindophenol☆

Automation of the glutathione peroxidase enzyme assay has been problematical. Although such methods have been reported, they do not give equivalent results to the standard manual assay, wherein glutathione oxidation is coupled to NADPH oxidation via glutathione reductase. We report here the development of a fully automated, continuous-flow, colorimetric method for glutathione peroxidase assays in which glutathione oxidation is monitored by its effect on the reaction of glutathione with the colorimetric reagent 2,6-dichloroindophenol. This method has a linear response to glutathione peroxidase over an 800-fold range of enzyme concentrations. Results of assays done by this method in erythrocyte and plasma samples correlate well with the standard manual coupled assay (r = 0.997 and 0.923, respectively), with no evidence of systematic errors. The assay works equally well with hydrogen peroxide or cumene hydroperoxide as substrate and shows the same selectivity toward glutathione S-transferases as the standard coupled assay. The within-day repeatability and the between-day reproducibility were estimated as 1.1 to 6.4% and 1.3 to 7.1% (relative standard deviation), respectively. This method is suitable for enzyme determinations in whole blood, erythrocytes, plasma, and serum from rats, rabbits, monkeys, and humans.