Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.     

Simultaneous HPLC analysis of 8-hydroxydeoxyguanosine and 7-methylguanine in urine from humans and rodents

With a recently developed high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, precise fraction collection, and reversed-phase chromatography, the oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OH-dG) was measured in human urine samples. The HPLC analysis was further modified to measure 8-OH-dG in rat and mouse urine samples. In addition, the urinary RNA degradation product 7-methylguanine (m7Gua) was analyzed simultaneously. The correlation coefficient (r) for the correlation between urinary creatinine and m7Gua was 0.9 for rats and 0.8 for humans and mice. Levels of 8-OH-dG in relation to urinary creatinine were compared and found to be similar for humans and rats and twice as high for mice. Urinary levels of m7Gua, as normalized to creatinine, were several-fold higher in rodents as compared with human levels, thereby correlating with the higher resting metabolic rate of rodents. The presented results show that 8-OH-dG and m7Gua can be analyzed simultaneously and reliably in urine from humans and rodents. In addition, m7Gua may be used as a reliable marker instead of creatinine for the normalization of 8-OH-dG in urine from rats and mice and also may be used in addition to normalization with creatinine in measurements of 8-OH-dG in human urine samples.     

The effect of Au injection on the ceruloplasmin,metallothionein and 8-hydroxydeoxyguanosine of rat serum,kidney and liver

The present study was carried out to investigate the effect of gold (Au) injection on copper (Cu) and two types of ceruloplasmin (Cp), total Cp (ID1) and active Cp (ID2), metallothionein (MT) in the serum, kidney and liver, and 8-hydroxydeoxyguanosine (8-OHdG) in the rat kidney. The Cu contents in sera and kidneys of Au-injected rats were 1.7 and 5.5 times higher than those in sera and kidneys of control rats, respectively. The most of Cu in the sera of the control rats or Au-injected rats were observed in the Cp fractions from a Sephacryl S-200 column. The Cu concentration in the Cp fractions was increased by Au injection. Significant increases of ID1 and ID2 were found in the sera of the control rats and Au-injected rats, while there was no significant difference in those concentrations of livers or kidneys between the control rats and Au-injected rats. Our results indicated that the most of Cp existed as active ID1. The immunoreactivity of 8-OHdG was located in the cortex of the Au-injected rat. These results indicated that the oxidative DNA damage occurred in the renal cortex of the Au-injected rat and the localization of DNA damage did not coincide with that of Cu–MT. These findings suggest that the oxidative DNA damage in the kidneys of rats injected with Au is associated with Cu except Cu–MT.     

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length)×50 μm I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 μg AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5–100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 μg AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.     

CFTR in K562 human leukemic cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.     

Glycophorins of human erythroleukemic K562 cells

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.     

Analysis of the oligosaccharides in ovalbumin by high-performance capillary electrophoresis

The oligosaccharides in ovalbumin as a glycoprotein model were released with anhydrous hydrazine, and reductively pyridylaminated after re-N-acetylation. The derivatives were analyzed by capillary zone electrophoresis (CZE) with on-column fluorometric detection. Direct CZE could separate the derivatives on the basis of the degree of polymerization, giving five peaks of hepta-, octa-, nona-, deca-, and undecasaccharides. Coelectrophoresis with the standard mixture of isomaltooligosaccharide derivatives was effective for peak assignment. CZE as borate complexes allowed separation on the basis of structural difference, especially in the peripheral monosaccharide residues. Peaks were tentatively assigned to the derivatives of reported oligosaccharides by comparing their relative mobilities with those of the chromatographic fractions obtained by using the ODS and Dowex 50W x 2 columns. These two modes gave excellent separation and were complementary to each other. Although the actual amount analyzed in the capillary tube was quite small (ca. 5 ng as carbohydrates), a larger amount (ca. 25 micrograms as carbohydrates) was required to make sample concentration sufficiently high to be detected by a modification of a commercial fluoromonitor for HPLC.     

No association between N7-methyldeoxyguanosine and 8-oxodeoxyguanosine levels in human lymphocyte DNA

To examine associations between two different classes of DNA damage that can occur through endogenous processes or exogenous exposures such as smoking, N7-methyldeoxyguanosine (N7-MedG) and 8-oxodeoxyguanosine (8-oxodG) levels were measured in lymphocyte DNA from 22 bronchoscopy patients. 8-OxodG and N7-MedG was detected in 100% and 91% of samples, respectively with 8-oxodG levels being approx 20 times higher (mean 8.39 ± 3.57 8-oxodG/10⁶dG versus 0.41 ± 0.33 N7-MedG/10⁶ dG) which provides an indication of the relative importance of the agents that induce oxidative DNA damage or alkylation damage. The sources of these genotoxic lesions remain to be established but N7-MedG and 8-oxodG levels were not correlated (r² < 0.01) suggesting that there is no association between alkylating agent and reactive oxygen species exposure, their metabolism and/or the DNA repair processes that can remove this DNA damage.     

Determination of the anticancer drug prospidin in human tissue by high-performance capillary electrophoresis using derivatization

A high-performance capillary electrophoresis (HPCE) method is described for the quantitative determination of the anticancer drug prospidin in human tissue after its derivatization with diethyldithiocarbamic acid sodium salt (DDTC). It was found that absorption of prospidin and its derivatives on the capillary wall due to the strong positive charge in the drug molecules could be eliminated by increasing the methanol content in the run buffer up to 50% and increasing the pH value up to 11.2. While studying the conditions of the interaction between prospidin and DDTC, a molar excess of the latter of 1:9 and 1.5 h of reaction time were found to be enough for complete derivatization. Sample preparation included homogenization of freshly cut papilloma species and deproteinization by methanol addition. Detection was by ultraviolet (UV) absorption at 254 nm. Due to its speed and high performance in separation. Detection was by ultraviolet (UV) absorption at 254 mm. Due to its speed and high practice.     

Induction of apoptosis in human leukemia K562 cells by cardiotoxin III

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III was found to inhibit the growth of K562 cells in a time-and dose-dependent manner with IC50 value of 1.7 microg/ml, and it displayed several features of apoptosis including apoptotic body formation, increase of sub G1 population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. Investigation of the mechanism of CTXIII--induced apoptosis revealed that the treatment of K562 cells with CTX III resulted in the activation of caspase-9, caspase-3 and subsequent cleavage of its substrate PARP and that CTXIII was also associated with an early release of cytochrome c from the mitochondria. These results suggest that CTX III may induce apoptosis through a mitochondria- and caspase-dependent mechanism.     

Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil–granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na₂HPO₄ buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na₂HPO₄ buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.     

8-hydroxydeoxyguanosine causes death of human leukemia cells deficient in 8-oxoguanine glycosylase 1 activity by inducing apoptosis

Our previous study showed that KG-1, a human acute leukemia cell line, has mutational loss of 8-oxoguanine (8-hydroxyguanine; oh(8)Gua) glycosylase 1 (OGG1) activity and that its viability is severely affected by 8-hydroxydeoxyguanosine (8-oxodeoxyguanosine; oh(8)dG). In the present study, the nature of the killing action of oh(8)dG on KG-1 was investigated. Signs observed in oh(8)dG-treated KG-1 cells indicated that death was due to apoptosis, as demonstrated by: increased sub-G(1) hypodiploid (apoptotic) cells, DNA fragmentation, and apoptotic body formation; loss of mitochondrial transmembrane potential, the release of cytochrome c from mitochondria into the cytosol, and the down-regulation of bcl-2; and the activation of caspases 8, 9, and 3, and the efficient inhibition of the apoptotic process by caspases inhibitors. This apoptosis appears not to be associated with Fas/Fas ligand because the expressions of these proteins were unchanged. Apoptotic KG-1 cells showed a high concentration of oh(8)Gua in DNA. Moreover, the increased concentration of oh(8)Gua in DNA, and the apoptotic process were not suppressed by the antioxidant, N-acetylcysteine, and thus the process is independent of reactive oxygen species. Of the 18 cancer cell lines treated with oh(8)dG, 3 cell lines (H9, CEM-CM3, and Molt-4) were found to be committed to apoptosis, and all of these showed very low OGG1 activity and a marked increase in the concentration of oh(8)Gua in DNA. These observations indicate that in addition to its mutagenic action, oh(8)Gua in DNA disturbs cell viability by inducing apoptosis.     

Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells

Using human erythroleukemia K562 cells, existence of receptors for hemopexin has been investigated. Hemopexin was bound to the cells in saturable, time- and temperature-dependent manner. The cells exhibited approximately 8,400 binding sites/cell for hemopexin and apohemopexin. The dissociation constants (Kd) for hemopexin and apohemopexin were 4.79 nM and 10.8 nM, respectively. Specific binding of labeled hemopexin was inhibited with increasing concentrations of unlabeled hemopexin and apohemopexin, but unaffected by transferrin and serum albumin. Heme bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that heme in hemopexin was taken up by K562 cells via the receptors for hemopexin.     

Biogenesis of glycophorin A in K562 human erythroleukemia cells

A monoclonal antibody (mAb-233) directed against an epitope in the nonglycosylated carboxyl-terminal region of human erythrocyte glycophorin A (GPA) was used in combination with metabolic labeling, the modification of N- and O-linked oligosaccharide processing by tunicamycin and monensin, and digestions with neuraminidase and O-glycanase to elucidate the pathway of GPA biogenesis in K562 human erythroleukemia cells. Cell-surface GPA is derived from two obligatory precursors in a stepwise manner. The initial GPA precursor has a Mr of 27,000 and appears to contain one N-linked high mannose oligosaccharide chain. In tunicamycin-treated cells, the initial precursor is similar in size (Mr = 24,000) to deglycosylated GPA from human erythrocytes. The 27-kDa initial precursor is rapidly converted to a transient 31-kDa intermediate by the addition of N-acetylgalactosamine residues to serine/threonine hydroxyl groups. Subsequent maturation involves the conversion of the high mannose chain to a complex-type oligosaccharide and the concomitant addition of galactose and sialic acid to internal N-acetylgalactosamine residues to extend the O-linked chains. These results define a single, stepwise processing pathway for the generation of all cell-surface GPA molecules and document for the first time the occurrence of both a unique initial precursor that contains a high mannose N-linked oligosaccharide chain but no O-linked sugars and a transient intermediate that appears to contain the same N-linked group and N-acetylgalactosamine at multiple serine/threonine residues. The properties of the intracellular GPA precursors and the relatively simple nature of the processing pathway reported herein contrast markedly with the characteristics of three intermediates and the complexity of two independent pathways in previously postulated schemes for GPA biogenesis (Gahmberg, C. G., Jokinen, M., Karhi, K. K., Kampe, O., Peterson, P. A., and Andersson, L. C. (1983) Methods Enzymol. 96, 281-298; Jokinen, M., Andersson, L. C., and Gahmberg, C. G. (1985) J. Biol. Chem. 260, 11314-11321).     

Biosynthetic regulation of the human transferrin receptor by desferrioxamine in K562 cells

Treatment of K562 cells with the iron chelator desferrioxamine results in the gradual increase in total cell receptors for transferrin. Receptor number rises 2.5-4.5-fold over 24 h and remains at the elevated level if the chelator is continuously present. Preincubation of the chelator with ferric chloride abolishes the effect. The drug has no effect on the 7-h half-life of the receptor. The increased number of receptors can be accounted for by a specific increase in the rate of receptor biosynthesis which reaches 3-4 times that seen in untreated cells by 6 h after the addition of the chelator. Isolation of mRNA from treated cells reveals that, after 8 h in the presence of desferrioxamine, there is a 3-fold increase in the specific translation of transferrin receptor over untreated cells. Total protein synthesis is not changed under these conditions.     

Erythroid induction of K562 human leukemia cells: enhancement by purines

K562 cells are human leukemia cells inducible for hemoglobin synthesis by a variety of agents. This report demonstrates that hypoxanthine, which alone has no inductive effect, enhances induction by thymidine, resulting in a greater absolute, as well as relative, percentage of benzidine positive cells. This effect is seen over a 20-fold concentration range for both thymidine and hypoxanthine. This enhancement involves commitment, i.e., a process in which the induction of hemoglobin synthesis is coupled to a limitation in the number of subsequent cell divisions. Although thymidine alone increases the percentage of cells in S phase, hypoxanthine does not augment this. Purines other than hypoxanthine also enhance induction by thymidine. This enhancement by hypoxanthine of thymidine induction is inhibited by pyrimidine nucleosides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, itself an effective K562 inducer, is not additive to thymidine and hypoxanthine, suggesting that hypoxanthine may act by reducing the supply of guanosine nucleosides.     

Determination of vitamin A in dried human blood spots by high-performance capillary electrophoresis with laser-excited fluorescence detection

We have developed a high-performance capillary electrophoresis (HPCE) method to analyze the retinol (vitamin A) concentration as retinol-retinol binding protein (holo-RBP) from microvolumes of serum (5–10 μl) or one to two drops (∼20 μl) of blood collected and air-dried on blood collection filter paper. A 0.64-cm diameter disk was cut from the dried whole blood specimens and the samples were dissolved in a pretreatment buffer and filtered. Filtrate was injected onto the HPCE column for analysis. The separation was carried out in a 60 cm × 50 μm I.D. fused-silica capillary and the running voltage was 20 kV. A HeCd laser with a wavelength of 325 nm was used for excitation, and the fluorescence of the holo-RBP complex was monitored at 465 nm by a photodiode. A virtual linear relationship was obtained for the retinol concentrations between HPCE and HPLC for 28 serum samples, 19 dried venous blood samples and 9 capillary dried blood spot samples, indicating that valid measures of serum retinol can be obtained from one to two drops of capillary blood collected on filter paper. The absolute detection limit for retinol by HPCE is below 3 μg/l. The method is very useful for vitamin A level screening, especially for children and premature new-born babies.     

Application of high-performance capillary electrophoresis to the purification process of Escherichia coli K4 polysaccharide

The high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) technique was applied to the extraction and purification process of the K4 polysaccharide from cultured bacteria in several stages. HPCE proved to be a technique with high resolution and sensitivity in analyzing K4 polysaccharide during its purification, in particular by using a strong anion-exchange resin. This is of paramount importance to monitor the product during the extraction and purification process or to test the purity of the final product. Furthermore, HPCE is able to verify that the extraction and purification process adopted is not carried out under drastic conditions capable of inducing fructose removal from the polysaccharide backbone.