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Xing-Fang Li

Publications and source records attributed to Xing-Fang Li.

At least 19 recordsLinked to original sources
PubMed2007

Binding of dimethylarsinous acid to cys-13alpha of rat hemoglobin is responsible for the retention of arsenic in rat blood.

The metabolism, disposition, and carcinogenicity of arsenic differ dramatically between humans and rats. To understand the molecular basis of these differences, we have characterized arsenic species in rats that were treated with inorganic arsenate (iAsV), monomethylarsonic acid (MMAV), or dimethylarsinic acid (DMAV) for up to 15 weeks. Arsenic significantly accumulated in the red blood cells (RBCs) of rats in the form of hemoglobin (Hb) complexed with dimethylarsinous acid (DMAIII), regardless of whether the rats were treated with iAsV, MMAV, or DMAV, suggesting rapid methylation of arsenic species followed by strong binding of DMAIII to rat Hb. The binding site for DMAIII was identified to be cysteine 13 in the alpha-chain of rat Hb with a stoichiometry of 1:1. Over 99% of the total arsenic (maximum 2.5-3.5 mM) in rat RBCs was bound to Hb for all rats examined (n = 138). In contrast, only 40-49% of the total arsenic (maximum approximately 10 muM) in rat plasma was bound to proteins. The ratios of the total arsenic in RBCs to that in plasma ranged from 88-423 for rats that were fed iAsV, 100-680 for rats that were fed MMAV, and 185-1393 for rats that were fed DMAV, when samples were obtained over the 15-week exposure duration. Previous studies have shown an increase in urothelial hyperplasia in rats fed DMAV. This is the first article reporting that treatment with iAsV in the drinking water also produces urothelial hyperplasia and at an even earlier time point than dietary DMAV. Dietary MMAV produced only a slight urothelial response. A correlation between the Hb-DMAIII complex and urothelial lesion severity in rats was observed. The lack of cysteine 13alpha in human Hb may be responsible for the shorter retention of arsenic in human blood. These differences in the disposition of arsenicals may contribute to the observed differences between humans and rats in susceptibility to arsenic carcinogenicity.

Amino Acid Sequence↗
PubMed2006

An electronic DNA microarray technique for detection and differentiation of viable Campylobacter species.

An electronic oligonucleotide microarray technique was developed for detection and differentiation of the viable Campylobacter species, C. jejuni, C. coli, and C. lari. This development consisted of four major components: identification of single nucleotide polymorphisms (SNPs) within the hsp60 gene as species markers, design of fluorescently labelled SNP-based reporters, development of an electronic microarray detection, and application of the integrated technique to analysis of Campylobacter species in food samples. A unique capability of this technique is the specific detection of viable cells and not dead ones. This is achieved by using mRNA of the 60 kDa heat-shock protein as the viability marker. The identification of two unique SNPs closely located at positions 291 and 294 of the hsp60 gene enabled the differentiation of the three Campylobacter species. This technique was able to detect as few as two viable Campylobacter cells. The analysis of 19 blind Campylobacter samples showed 100% agreement with their identities obtained using pulsed-field gel electrophoresis. The analysis of six chicken samples revealed the presence of C. coli in one of the samples.

Animals↗
PubMed2006

Top-down characterization of proteins and drug-protein complexes using nanoelectrospray tandem mass spectrometry.

We report a 'top-down' approach for characterization of proteins, and identification of binding sites in protein-drug complexes using nanoelectrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry (nanoESI-MS/MS). The efficiency of direct fragmentation of intact protein ions and the feasibility of this method were initially demonstrated using several well-characterized proteins with different molecular weights including metallothionein (6126 Da), cytochrome c (horse, 12360 Da), myoglobin (horse, 16592 Da), and hemoglobin (human, 64453 Da). Simply varying collision energy without enzyme digestion and gel or LC separation generated a range of peptide fragments of these proteins. Over 80% of these peptide ions matched those in the SWISS-PROT database with mass accuracy of 8 to 32 ppm with external calibration. This technique was further applied to fragment a cisplatin-metallothionein complex to identify the binding sites, demonstrating a potential application in the study of drug-protein binding.

Amino Acid Sequence↗
PubMed2006

Mass spectrometry study of hemoglobin-oxaliplatin complexes in colorectal cancer patients and potential association with chemotherapeutic responses.

Oxaliplatin is the most active platinum (Pt)-containing anticancer drug for the treatment of advanced colorectal cancer. We report here the study of potential association of the levels of oxaliplatin-protein complexes in 19 cancer patients with treatment efficacy using size-exclusion high-performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC/ICPMS) and nanoelectrospray ionization mass spectrometry (nanoESI-MS) techniques. Blood samples from 19 colorectal cancer patients were collected at 1 and 48 h after the first infusion of oxaliplatin. HPLC/ICPMS quantification of the oxaliplatin-protein complexes showed that the levels of Pt-protein complexes in plasma samples at 48 h were reduced by approximately 50% compared to those at 1 h, whereas those in hemolysates did not change significantly. The concentrations of hemoglobin (Hb)-oxaliplatin complexes determined by HPLC/ICPMS ranged from 3.1 to 8.7 microM. NanoESI-MS analysis of the patient hemolysates showed three distinct mass spectral profiles of the Hb-oxaliplatin complexes: (1) 1:1, (2) 1:1 with 1:2, and (3) multiple complexes of 1:1, 1:2, 1:3, and 1:4, corresponding to the Hb-oxaliplatin complex concentrations determined by HPLC/ICPMS. Potential association of variables including Hb-oxaliplatin complex concentrations with time to progress as the treatment efficacy indicator was analyzed using the Cox model. Multivariate analysis of the potential predictors showed that the statistically significant variables were Hb-oxaliplatin complex concentration (p = 0.02), performance status (p = 0.02), baseline neutrophil count (p = 0.05), and the site of the primary cancer (colon vs. rectal, p = 0.01). The hazard ratio for the concentration of the Hb-oxaliplatin complexes was 2.4, suggesting that the risk of cancer progression significantly increased with increasing of Hb-oxaliplatin complexes in patients. These results demonstrate that the level of the Hb-oxaliplatin complexes in erythrocytes is a potential biomarker for indicating inter-patient variations in oxaliplatin treatment efficacy.

Adenocarcinoma↗
PubMed2006

Determination of microcystins in water using integrated solid-phase microextraction with microbore high-performance liquid chromatography-electrospray quadruple time-of-flight mass spectrometry.

The development of a technique combining solid-phase microextraction (SPME) with microbore high-performance liquid chromatography (micro-HPLC)-tandem quadrupole time-of-flight (QTOF) mass spectrometry (MS) for determination of dissolved microcystins in water is reported. Several important parameters affecting the efficiency of SPME extraction of microcystins are investigated. A microbore C18 column HPLC coupled with tandem QTOF-MS with information-dependent acquisition (IDA) is developed to effectively analyze microcystins in microliter volumes of SPME extracts. The micro-HPLC-QTOF-MS with IDA technique provides comprehensive information, including a survey chromatogram (total ion chromatogram), full scan mass spectrum, and product ion scan mass spectra at different collision energies for individual analytes, which allows for both identification and quantitation in the same run. Linear calibration curves of microcystin standard [microcystin (MC)-arginine (R)R] 1-100 microg/L and of microcystin standard [MC-leucine (L)R] 1-250 microg/L are obtained with a correlation coefficient of 0.996. The combination of SPME with HPLC-QTOF-MS and IDA offers limits of detection of 0.6 pg for MC-RR and 1.6 pg for MC-LR. Analysis of spiked lake-water samples shows a recovery of > 86% for MC-RR and > 70% for MC-LR. This technique requires small sample volumes, minimizes the use of organic solvents, and provides sensitive and information-rich analysis of unknown samples.

Chromatography, High Pressure Liquid↗
PubMed2006

Chromium on the hands of children after playing in playgrounds built from chromated copper arsenate (CCA)-treated wood.

Children's exposure to arsenic and chromium from playground equipment constructed with chromated copper arsenate (CCA)-treated wood is a potential concern because of children's hand-to-mouth activity. However, there exists no direct measure of Cr levels on the hands of children after playing in such playgrounds. In this study we measured both soluble and total Cr on the hands of 139 children playing in playgrounds, eight of which were constructed with CCA-treated wood and eight of which were not. Children's age and duration of play were recorded. The hands of each child were washed after play with 150 mL deionized water, which was collected in a bag and subsequently underwent analysis of Cr and 20 other elements, using inductively coupled plasma mass spectrometry. Total average Cr on the hands of 63 children who played in CCA playgrounds was 1,112 +/- 1,089 ng (median, 688; range 78-5,875). Total average Cr on the hands of 64 children who played in non-CCA playgrounds was 652 +/- 586 ng (median, 492; range 61-3,377). The difference between the two groups is statistically significant (p < 0.01). Cr levels were highly correlated to both Cu (r = 0.672) and As (r = 0.736) levels in CCA playgrounds (p < or = 0.01), but not non-CCA playgrounds (r = 0.252 and 0.486 for Cu and As, respectively). Principal-component analysis indicates that Cr, Cu, and As are more closely grouped together in CCA than in non-CCA playgrounds. These results suggest that the elevated levels of Cr and As on children's hands are due to direct contact with CCA wood.

Arsenates↗
PubMed2005

Characterization of intact hemoglobin and oxaliplatin interaction by nanoelectrospray ionization tandem mass spectrometry.

BACKGROUND: Mass spectrometric (MS) detection of intact hemoglobin (Hb) adducts presents considerable analytical challenges because of the noncovalent association of the 4 subunits of Hb, and MS characterization of the interaction of intact Hb with platinum drugs has not been reported. We developed a technique for detecting intact Hb and its drug adduct and studied the interactions between intact Hb and oxaliplatin. METHODS: We incubated a series of mixtures of Hb and oxaliplatin at 37 degrees C for 24 h or 5 days to investigate adduct formation. Blood samples from colorectal cancer patients undergoing oxaliplatin treatment were analyzed for novel adducts of intact Hb-oxaliplatin, which were characterized with nanoelectrospray ionization quadrupole time-of-flight MS. RESULTS: Two intact Hb adducts, one with the whole oxaliplatin molecule and the other with oxaliplatin losing the oxalate ligand, were identified. Analysis of erythrocytes from the cancer patients provided direct evidence that oxaliplatin accumulated as Hb adducts in erythrocytes. A higher fraction (approximately 70%) of Hb was bound to oxaliplatin in erythrocytes from a patient who could not tolerate oxaliplatin treatment than in erythrocytes from another patient who benefited from this treatment. CONCLUSIONS: The nanoelectrospray tandem MS technique enabled determination of the intact Hb tetramer and its association with oxaliplatin. Hb-oxaliplatin adducts in erythrocytes may serve as a clinical biomarker for toxic response and treatment efficacy.

Colorectal Neoplasms↗
PubMed2005

Detection of fortification of ginkgo products using nanoelectrospray ionization mass spectrometry.

We report here a negative ionization nanoelectrospray ionization mass spectrometry (nanoESI-MS) technique that simultaneously detects active components, terpenes and intact flavonol glycosides, and toxic ginkgolic acids in ginkgo products. Unlike the conventional methods that hydrolyze flavonol glycosides to flavonoids for analysis, this technique directly detects intact flavonol glycosides, enabling differentiation of these natural glycosides from the synthetic flavonoids. Thus, it allows the detection of fortification of ginkgo products, alleviating a common problem encountered by the conventional methods. Analysis of 14 commercial ginkgo products using this technique demonstrates large variations and deviation from the well-accepted standardized ginkgo extract. Four products showed evidence of fortification with synthetic surrogates. Two products were found to have toxic ginkgolic acids that exceed the 5 microg g(-1) limit by as much as 60000 fold. These results emphasize the importance of appropriate monitoring of ginkgo product quality.

Drug Contamination↗
PubMed2005

Intact human holo-transferrin interaction with oxaliplatin.

We report the interaction of intact human holo-transferrin (holo-Tf) with oxaliplatin (an anticancer drug), and the characterization of a complex composed of (1:1) intact holo-Tf and the parent oxaliplatin molecule using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS). The molecular weight of this complex was determined to be 80,077 Da, which was an increase of 397 mass units compared to the protein alone (79,680 Da), suggesting that a parent drug molecule was bound to the intact protein. We further examined the interaction between the intact protein and oxaliplatin using size-exclusion high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS). The protein complex and free oxaliplatin were separated by HPLC and quantitatively determined by simultaneous monitoring of both 195Pt and 56Fe using ICPMS. The HPLC/ICPMS detected both Pt and Fe signals at retention time of 2.6 min, identifying the protein-drug complex. The Fe signal at 2.6 min did not change with the increase in incubation time of the reaction mixture containing holo-Tf and oxaliplatin, while the Pt signal at the same retention time increased over time, further demonstrating that the formation of this complex does not affect the protein-bound Fe. The binding constant of the (1:1) intact human holo-Tf-oxaliplatin complex was determined to be 7.7x10(5) M-1. Both nanoESI-MS and HPLC/ICPMS results support that the holo-Tf and parent oxaliplatin molecules form complexes through non-covalent binding, suggesting that holo-Tf may be a useful carrier for oxaliplatin delivery.

Antineoplastic Agents↗
PubMed2004

Reverse transcription-multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi.

BACKGROUND: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens. METHODS: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates. RESULTS: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis. CONCLUSION: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.

Bacterial Proteins↗
PubMed2004

Evidence of hemoglobin binding to arsenic as a basis for the accumulation of arsenic in rat blood.

Four trivalent arsenic species, inorganic arsenite (iAs(III)), monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and phenylarsine oxide (PhAs(III)O), have shown increasing binding affinity with the hemoglobin (Hb) of rats and humans. The binding stoichiometry was consistent with the number of reactive cysteine residues in the alpha and beta chains of Hb. Comparing the binding affinity of rat Hb and human Hb for the same trivalent arsenic species, rat Hb was 3-16 times stronger than human Hb as demonstrated by their apparent binding constants. Comparative experiments involving incubation of human and rat red blood cells (RBC) with iAs(III), MMA(III), and DMA(III) showed that 15-30-fold more arsenic species were bound to the Hb of rat RBC than that of human RBC. In vivo experiments using rats fed with an arsenic-supplemented diet showed that arsenic in RBC of the rats was predominantly found in the protein-bound form. Further characterization by nanoelectrospray mass spectrometry of the arsenic species in the RBC of these rats confirmed that most arsenic was bound to the alpha chain of Hb. Taken together, these results suggest that the stronger binding affinity of these arsenic species to rat Hb is responsible for the accumulation of arsenic in rat blood. The results provide a chemical basis to explain the previously observed intriguing difference in the retention of arsenic in the human and the rat. The techniques and approaches described can be applied to the studies of arsenic interactions with other functional proteins.

Animals↗
PubMed2004

Interaction of oxaliplatin, cisplatin, and carboplatin with hemoglobin and the resulting release of a heme group.

Oxaliplatin, carboplatin, and cisplatin are widely used to treat a number of cancers. While their DNA adducts are believed to cause cell death, the involvement of their protein adducts in the toxicity and action of these drugs is unclear. Here, we report the interactions of hemoglobin (Hb) with the three platinum (Pt) drugs, demonstrating the formation of Hb-Pt complexes and the release of a heme group from Hb. Oxaliplatin (0.05 microM) was able to form three major complexes with Hb (3-10 microM) after 1 h of incubation at room temperature, and these complexes accounted for approximately 60% of the total oxaliplatin. Cisplatin and carboplatin formed one major and two minor complexes only after 24 and 96 h of incubation, respectively. Incubation of these Pt drugs (0.05-10 microM) with whole blood of healthy volunteers and the analysis of red blood cells confirmed the relative ability of these Pt drugs binding to Hb. For the whole blood samples incubated with oxaliplatin and cisplatin for 24 h, only protein complexes were detected in red blood cells, indicating a complete binding of oxaliplatin and cisplatin to the protein. In contrast, carboplatin was partially bound; both the free and the protein-bound carboplatin species were detected in red blood cells. The binding of the Pt drugs to Hb was accompanied by the release of a heme group from Hb, which was monitored by size fractionation, chromatographic separation, and selective detection of both Pt- and iron (Fe)-containing molecular species. The released heme was further identified by size fractionation and nanospray mass spectrometry. The findings of the Pt drug interaction with Hb and the dissociation of heme from Hb are potentially useful for a better understanding of the toxicity and side effects of these chemotherapeutic drugs.

Binding, Competitive↗
PubMed2004

Arsenic on the hands of children after playing in playgrounds.

Increasing concerns over the use of wood treated with chromated copper arsenate (CCA) in playground structures arise from potential exposure to arsenic of children playing in these playgrounds. Limited data from previous studies analyzing arsenic levels in sand samples collected from CCA playgrounds are inconsistent and cannot be directly translated to the amount of children's exposure to arsenic. The objective of this study was to determine the quantitative amounts of arsenic on the hands of children in contact with CCA-treated wood structures or sand in playgrounds. We compared arsenic levels on the hands of 66 children playing in eight CCA playgrounds with levels of arsenic found on the hands of 64 children playing in another eight playgrounds not constructed with CCA-treated wood. The children's age and duration of playtime were recorded at each playground. After play, children's hands were washed in a bag containing 150 mL of deionized water. Arsenic levels in the hand-washing water were quantified by inductively coupled plasma mass spectrometry. Our results show that the ages of the children sampled and the duration of play in the playgrounds were similar between the groups of CCA and non-CCA playgrounds. The mean amount of water-soluble arsenic on children's hands from CCA playgrounds was 0.50 microg (range, 0.0078-3.5 microg). This was significantly higher (p < 0.001) than the mean amount of water-soluble arsenic on children's hands from non-CCA playgrounds, which was 0.095 microg (range, 0.011-0.41 microg). There was no significant difference in the amount of sand on the children's hands and the concentration of arsenic in the sand between the CCA and non-CCA groups. The higher values of arsenic on the hands of children playing in the CCA playgrounds are probably due to direct contact with CCA-treated wood. Washing hands after play would reduce the levels of potential exposure because most of the arsenic on children's hands was washed off with water. The maximum amount of arsenic on children's hands from the entire group of study participants was < 4 microg, which is lower than the average daily intake of arsenic from water and food.

Arsenates↗
PubMed2003

Mass spectrometric studies of cisplatin-induced changes of hemoglobin.

This study reports on structural changes of hemoglobin (Hb) that were induced by cisplatin binding. Two techniques, nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoES-MS) and high-performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC/ICPMS), were developed to facilitate this study. Nanospray MS analyses of cisplatin and Hb reaction mixtures demonstrated that the ion at m/z 616.5, the heme group, increased with an increase of cisplatin concentration, indicating the loss of heme groups from the intact protein. This conclusion was also supported by the increase of cisplatin-alpha or -beta complex formation. The change of the Hb-bound Fe was further investigated by monitoring Fe signals using size-exclusion HPLC/ICPMS. After incubation with cisplatin at clinically relevant concentrations, under physiological conditions, the amount of Fe bound to Hb was reduced while formation of cisplatin-Hb complexes increased. Flow-injection ICPMS analysis of the Fe contents in the low molecular weight fraction (<3000 Da) of the reaction mixtures after size fractionation further demonstrated a corresponding increase of Fe with the increase of cisplatin concentrations. HPLC/ICPMS detected three Hb-cisplatin complexes, one of which eluted at the same retention time as Hb while the other two complexes eluted later than Hb. With clinically relevant concentrations of cisplatin (0.05-1.0 microM) and 10 microM of Hb, the concentrations of the Hb-cisplatin complexes were determined in the range 0.1-64 nM. These results, obtained from nanoES-MS, HPLC/ICPMS, and FIA-ICPMS, demonstrate that cisplatin binding to Hb resulted in the dissociation of the heme group from the intact protein.

Binding Sites↗
PubMed2003

Interaction of trivalent arsenicals with metallothionein.

Arsenic is a human carcinogen, causing skin, bladder, and lung cancers. Although arsenic in drinking water affects millions of people worldwide, the mechanism(s) of action by which arsenic causes cancers is not known. Arsenic probably exerts some toxic effects by binding with proteins. However, few experimental data are available on arsenic-containing proteins in biological systems. This study reports on arsenic interaction with metallothionein and established binding stoichiometries between metallothionein and the recently discovered trivalent metabolites of arsenic metabolism. Size exclusion chromatography with inductively coupled plasma mass spectrometry analysis of reaction mixtures between trivalent arsenicals and metallothionein clearly demonstrated the formation of complexes of arsenic with metallothionein. Analysis of the complexes using electrospray quadrupole time-of-flight tandem mass spectrometry revealed the detailed binding stoichiometry between arsenic and the 20 Cys residues in the metallothionein molecule. Inorganic arsenite (As(III)) and its two trivalent methylation metabolites, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), readily bind with metallothionein. Each metallothionein molecule could bind with up to six As(III), 10 MMA(III), and 20 DMA(III) molecules, consistent with the coordination chemistry of these arsenicals. The findings on arsenic interaction with proteins are useful for a better understanding of arsenic health effects.

Arsenic↗