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S Naaby-Hansen,
M J Wolkowicz,
K Klotz,
L A Bush,
V A Westbrook,
H Shibahara,
J Shetty,
S A Coonrod,
P P Reddi,
J Shannon, M Kinter,
N E Sherman,
J Fox,
C J Flickinger,
J C Herr
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ABSTRACT: Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a (45)Ca overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP(3) receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.
Molecular Human Reproduction 11/2001; 7(10):923-33. · 3.85 Impact Factor
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ABSTRACT: Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.
AJP Lung Cellular and Molecular Physiology 11/2000; 279(4):L716-21. · 3.66 Impact Factor
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A Mandal,
S Naaby-Hansen,
M J Wolkowicz,
K Klotz,
J Shetty,
J D Retief,
S A Coonrod, M Kinter,
N Sherman,
F Cesar,
C J Flickinger,
J C Herr
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ABSTRACT: Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.
Biology of Reproduction 12/1999; 61(5):1184-97. · 4.01 Impact Factor
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ABSTRACT: Obstructive nephropathy leads to progressive renal tubular atrophy and interstitial fibrosis and is associated with sodium wasting and sodium depletion. Renal damage resulting from unilateral ureteral obstruction (UUO) may be aggravated by reactive oxygen species (ROS), which are produced by a variety of processes. Ideally, deleterious effects of ROS are attenuated by antioxidant enzymes, including the superoxide dismutases, glutathione peroxidases, catalase, and glutathione-S-transferases. The general paradigm is that tissue damage occurs when ROS production is greater than the protective capacity of the antioxidant enzymes.
This study was designed to investigate the response of renal antioxidant enzymes to UUO and sodium depletion. Adult, male Sprague-Dawley rats received normal-sodium or sodium-depleted siets and were subjected to UUO or sham operation. Obstructed (UUO), intact opposite, or sham-operated kidneys were harvested after 14 days, and antioxidant enzyme activities were measured in kidney homogenates. Thiobarbituric acid reactive substances were measured in these homogenates at 3 and 14 days after UUO or sham operation as an index of ROS production.
Renal interstitial area, a measure of fibrosis, was increased by UUO and was doubled in sodium-depleted animals. Sodium depletion increased manganese superoxide dismutase, glutathione peroxidases, and glutathione-S-transferase activities in sham-operated kidneys but not in UUO kidneys. Relative to intact opposite kidneys, UUO kidneys had reduced activities of catalase, manganese superoxide dismutase, and glutathione-S-transferase in normal-sodium animals and all antioxidant enzymes tested in sodium-depleted animals. Renal thiobarbituric acid reactive substances were increased by three days of UUO and were increased further by 14 days of sodium depletion.
In summary, sodium depletion increased several renal antioxidant enzymes, consistent with a stress response to increased ROS production. Further, UUO not only reduced antioxidant enzyme activities but also inhibited increases seen with sodium depletion. We conclude that suppression of renal antioxidant enzyme activities by UUO contributes to the progression of renal injury in obstructive nephropathy, a process exacerbated by sodium depletion.
Kidney International 05/1999; 55(4):1327-34. · 6.61 Impact Factor
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ABSTRACT: p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.
Molecular and Cellular Biology 01/1999; 18(12):7052-63. · 5.53 Impact Factor
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ABSTRACT: The effects of oleic, linoleic and arachidonic acid on oxygen toxicity were evaluated in cultured hamster fibroblasts. Each fatty acid was incorporated separately using a protocol that resulted in dose-dependent increases in the respective cellular fatty acid content to as much as 20-fold greater than unsupplemented controls. Linoleic acid produced no changes in cell survival after 48 h treatment with 95% oxygen, regardless of fatty acid content of the cells. Oleic acid incorporation resulted in a dose-dependent increase in cell survival at 48 h in 95% oxygen, whereas arachidonic acid incorporation resulted in a dose-dependent decrease in cell survival at 48 h in 95% oxygen. No significant differences in amounts of linoleic or arachidonic acids were detected in control, oleic acid- enriched or linoleic acid-enriched cells during oxygen exposure. In cells enriched with arachidonic acid, exposure to oxygen significantly reduced the amounts of linoleic and arachidonic acid to 79 and 84%, respectively, of the amounts found in air-exposed cells. The results indicate that oleic acid incorporation into cells provides protection against 95% oxygen-induced cytotoxicity. In contrast, arachidonic acid incorporation led to sensitization of cells to 95% oxygen-induced cytotoxicity that was accompanied by a loss of polyunsaturated fatty acids. As a result, it would appear that in situations of increased oxidative stress, high monounsaturated fatty acid diets that increase cellular oleic acid content may provide a protective environment compared with high polyunsaturated fatty acid diets that increase cellular arachidonic acid content.
Journal of Nutrition 01/1997; 126(12):2952-9. · 3.92 Impact Factor
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ABSTRACT: Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1,4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1,4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.
Free Radical Research 08/1996; 25(1):23-9. · 2.88 Impact Factor
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ABSTRACT: Treatment of cultured fibroblasts, designated HA1 cells, with 4-hydroxy-2-nonenal (4HNE) in doses up to 50 nmol/10(6) cells for 3 h results in dose-dependent cytotoxicity measured by clonogenic cell survival with 50% cytotoxicity achieved at 32 nmol 4HNE/10(6) cells. 4HNE treatment also resulted in dose-dependent reduction of cellular glutathione (GSH) content and loss of glutathione peroxidase (GPx) activity at 4HNE doses greater than 15 nmol/10(6) cells. By comparison, a 95% oxygen-resistant variant of HA1 cells, designated O2R95 cells, and a hydrogen peroxide-resistant variant of HA1 cells, designated OC14 cells, were found resistant to 4HNE cytotoxicity requiring 54 nmol 4HNE/10(6) cells and 75 nmol 4HNE/10(6) cells, respectively, for 50% cytotoxicity. In O2R95 cells, dose-dependent decreases were seen in GSH levels and GPx activity. In OC14 cells, however, any reduction in cellular GSH levels required doses of 4HNE greater than 30 nmol/10(6) cells, and GPx activity remained unchanged. No changes were seen in glutathione-S-transferase activity in any of the cell lines at any dose tested. These data indicate a correlation between glutathione modification, in a manner that prevents its recycling, the ability to inactivate enzymes with active site selenocysteine residues and the cytotoxicity of alpha, beta-unsaturated aldehydes such as 4HNE.
Free Radical Biology and Medicine 02/1996; 21(4):457-62. · 5.42 Impact Factor
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M Kinter
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ABSTRACT: Productive investigation of the contribution of oxidative stress to human disease is facilitated by the design and application of suitable analytical technologies for oxidation product analysis. Lipid oxidation, including polyunsaturated fatty acid and cholesterol oxidation, produces a variety of products that can function as indexes of the extent of oxidation. These products include fatty acid hydroperoxides and hydroxides, aldehydes, prostanoids, hydrocarbons, and cholesterol hydroperoxides and hydroxides, epoxides, and carbonyls. Some of these oxidation products have biological activities that can contribute to tissue damage in unique ways. This paper reviews the state-of-the-art for chromatographic analysis of these products through a discussion of advances that have taken place since 1990.
Journal of chromatography. B, Biomedical applications 10/1995; 671(1-2):223-36.
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ABSTRACT: A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) method is described for the determination of lead (Pb) in urine and whole blood. The use of lithium bis(trifluoroethyl)dithiocarbamate Pb(FDEDTC) as a chelating agent showed strong memory effect, restricting the range of Pb isotope ratios that can be measured in unknown samples. To overcome this carryover problem, we further derivatized the Pb(FDEDTC)2 chelate with 4-fluorophenyl magnesium bromide to form Pb(FC6H4)4. The sequential analyses of solutions of natural Pb and enriched 204Pb with Pb(FC6H4)4 chelate by GC-MS demonstrated no observable memory effect. Precision and accuracy of Pb isotope ratio measurements with Pb(FC6H4)4 were established, and the isotope dilution GC-MS method was validated by determining Pb concentrations in urine standards from the National Institute of Standards and Technology, urine and blood reference materials from the New York State Department of Health, and blood Pb survey samples from the College of American Pathologists.
Clinical Chemistry 09/1994; 40(8):1494-502. · 7.91 Impact Factor
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ABSTRACT: The antitumor drug AS-101 [ammoniumtrichloro (dioxoethylene-O,O')tellurate(IV)] is the first tellurium-containing compound that has been identified as possessing immunomodulating properties and minimal toxicity. We have developed a stable isotope dilution gas chromatography/mass spectrometry method using 120Te as an internal standard and (4-fluorophenyl)magnesium bromide as a derivatizing agent for Te determination in urine. The urine samples were digested using HNO3 + H2O2 prior to derivatization with lithium bis(trifluoroethyl)dithiocarbamate at a pH of 3. The trifluorodiethyldithiocarbamate of tellurium was reacted with the Grignard reagent in anhydrous diethyl ether to obtain Te-(FC6H4)2 for GC/MS analysis. All isotope ratio measurements were made by selected ion monitoring with a Finnigan MAT 8230 organic mass spectrometer using a 10-m fused silica capillary column. Overall percision values for the five major Te isotopes relative to 130Te were 0.6-3.1% when 10-ng samples of chelated Te were analyzed. No appreciable memory or carry-over effect was observed when two synthetic mixtures differing in 120Te:130Te ratios by a factor of 50 were sequentially analyzed. The isotope dilution GC/MS method was validated by determining Te in urine samples and comparing the values with electrothermal atomic absorption spectrometry. Te concentrations were determined in the 100-500 micrograms/L range with CVs of 1-4%.
Analytical Chemistry 05/1994; 66(8):1316-22. · 5.86 Impact Factor
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ABSTRACT: A stable isotope dilution gas chromatography-mass spectrometry method using 196Hg as an internal standard is described for determining Hg in blood. In this method, the blood samples are not subjected to any digestion to avoid the loss of Hg. A solution of 0.6M HCl is used to free Hg present in blood from proteins. The pH of the solution is adjusted to 9 using borate buffer and Hg chelated using lithium bis(trifluoroethyl)dithiocarbamate. All isotope ratio measurements are made using an organic mass spectrometer. Overall precision values for the five major Hg isotopes relative to 202Hg are 1.6-2.3% when 10 ng samples of chelated Hg are analyzed. No appreciable memory or carryover effect is observed when two synthetic mixtures differing in 196Hg/202Hg ratios by a factor of 30 are sequentially analyzed. The method is validated by determining Hg in blood samples using isotope dilution GC-MS.
Biological Trace Element Research 04/1994; 41(1-2):89-102. · 1.92 Impact Factor
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ABSTRACT: Both plasma and whole blood concentrations of 4-hydroxy-2-nonenal (4HNE) were significantly elevated in a population (n = 6) of 2 year old Watanabe heritable hyperlipidemic rabbits relative to a population (n = 6) of New Zealand White rabbits. The plasma concentrations were 74 +/- 10 nmol/L for the Watanabe group and 47 +/- 6 nmol/L for the New Zealand White group. The whole blood concentrations were 364 +/- 55 nmol/L for the Watanabe group and 188 +/- 64 nmol/L for the New Zealand White group. These results indicate that 4HNE concentrations in blood can be elevated in individuals with atherosclerosis and demonstrate the potential link between the formation of 4HNE and the progression of atherosclerosis.
Biochemical and Biophysical Research Communications 04/1994; 199(2):671-5. · 2.48 Impact Factor
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Transactions of the American Clinical and Climatological Association 02/1994; 105:120-9; discussion 130.
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ABSTRACT: Mass spectrometry is a powerful analytical tool for determining the isotope ratios and concentrations of trace elements in various samples at levels ranging from major constituents to subparts per billion. Because isotope dilution is free from matrix effects, it has the potential of being incorporated into a definitive analytical approach that can provide reference values for concentrations in physiological and pathological conditions. In addition, isotope dilution mass spectrometry results are free from the constraints of quantitative recovery of the analyte, an essential requirement in other analytical techniques that is difficult to achieve with complex biological samples. A variety of mass spectrometric approaches have been used for determining the concentration of trace elements in biological samples. The more commonly used are thermal ionization mass spectrometry, inductively coupled plasma mass spectrometry, fast atom bombardment mass spectrometry, and gas chromatography mass spectrometry. This article reviews the work on trace element determination in biological samples using different mass spectrometric techniques and highlights the experiments performed by the authors in establishing gas chromatography mass spectrometry.
Critical Reviews in Clinical Laboratory Sciences 02/1994; 31(1):35-87. · 5.25 Impact Factor
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ABSTRACT: Isotope dilution gas chromatography-mass spectrometry (GC-MS) and electrothermal atomic absorption spectrometry (EAAS) are compared for platinum (Pt) determination in urine, plasma ultrafiltrate, and plasma samples from a patient undergoing cisplatin therapy. The isotope dilution GC-MS method is based on the use of lithium bis(trifluoroethyl)dithiocarbamate as a chelating agent and enriched 192Pt as an internal standard. Pt isotope ratios were measured using a Finnigan MAT 8230 organic mass spectrometer, and Pt concentrations were calculated from different sets of isotope ratios in the molecular ion of the Pt-chelate. In the EAAS method, Pt concentrations were determined using three different approaches. These were: (i) calibration curve based on aqueous standards containing Pt in 10% HCl, (ii) standard addition, and (iii) matrix digestion followed by standard addition. Good agreement was obtained for Pt concentrations determined by GC-MS and EAAS in urine samples while there were significant differences in Pt concentrations of ultrafiltrate and whole plasma samples by the two methods. Discussion of possible reasons for these differences emphasizes the need for future critical evaluation of these methods.
Analytical Biochemistry 05/1993; 210(1):113-8. · 3.00 Impact Factor
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ABSTRACT: A gas chromatographic-mass spectrometric method for the determination of the lipid aldehyde 4-hydroxy-2-nonenal (4HNE) in trace quantities is described. The method utilizes the reaction of aldehydes with hydroxylamine leading to the formation of the oxime derivative. The aldehydes are recovered by octadecylsilyl solid-phase extraction and converted to the bis-tert.-butyldimethylsilyl derivatives for analysis using electron ionization. A novel 4HNE analogue, 3-hydroxynonanal, has been synthesized and is used as an internal standard. A limit of detection of approximately 1 pmol of 4 HNE in preparations of approximately 2.10(6) cells or 0.5 ml of whole blood, plasma or serum was observed. Standard addition analysis indicates that the method is accurate at these levels. Replicate analysis of the National Institutes of Standards and Technology Standard Reference Material SRM 909 indicates an average in-run precision of 8.1% and a between-run precision of 13.5% at an average concentration of 82.1 pmol/ml of reconstituted material.
Journal of Chromatography 08/1992; 578(1):9-16. · 4.53 Impact Factor
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ABSTRACT: An isotope dilution gas chromatography-mass spectrometry method for selenium determination in urine using 76Se as an internal standard is described. Three different derivatizing reagents, 4-nitro-o-phenylenediamine (NPD), 3,5-dibromo-o-phenylenediamine (DBPD), and 4-trifluoromethyl-o-phenylenediamine (TFMPD) were investigated for their gas chromatographic behavior including memory and precision and accuracy of isotope ratio measurements. By these criteria, the performance of these reagents was TFMPD greater than DBPD greater than NPD. Overall precision values of 1 to 7% were observed in determining Se isotope ratios at the 10-ng level, with no significant difference in using any of the three reagents. Memory effect was observed in the order NPD greater than DBPD greater than TFMPD with TFMPD showing no measurable memory effect. Accuracy of the GC-MS method was verified by quantitation of selenium in the NIST freeze-dried urine reference material SRM 2670.
Analytical Biochemistry 06/1992; 202(2):367-74. · 3.00 Impact Factor
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ABSTRACT: A gas chromatographic-mass spectrometric method for the determination of cobalt in biological materials employing stable enriched 62Ni as an internal standard and using lithium bis(trifluoroethyl)dithiocarbamate as a chelating agent is described. The method involves the addition of a known amount (1 microgram) of 62Ni to the sample, the formation of the chelate and the determination by selected-ion monitoring of the m/z 571/574 ratio, which corresponds to 59Co/62Ni. No appreciable memory effect was observed, and an acceptable dynamic range of 100 was found. There was good agreement between the cobalt concentration values determined by gas chromatography-mass spectrometry and electrothermal atomic absorption spectrometry. The present method has high sensitivity and can be used for the quantitation of cobalt at concentrations as low as 1 microgram/l. The use of enriched 62Ni circumvents the problem caused by endogenous nickel and simultaneously provides data on the nickel concentration in the biological sample without any additional experimental effort.
Journal of Chromatography 06/1992; 576(2):297-304. · 4.53 Impact Factor
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ABSTRACT: A stable isotope dilution gas chromatography-mass spectrometry method using enriched 65Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N'-ethylenebis-(trifluoroacetylacetoneimine) [H2(enTFA2)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H2(enTFA2) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a 10-ng level using H2(enTFA2). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H2(enTFA2) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data.
Analytical Biochemistry 05/1991; 194(1):140-5. · 3.00 Impact Factor
