Biomedical subjects

B A Buchholz

Publications and source records attributed to B A Buchholz.

13 recordsLinked to original sources

PubMed2001

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry.

Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of (14)C. The transgenic transferase was labeled by growing transformed Escherichia coli on [(14)C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.

Carbon Radioisotopes↗

PubMed2001

Microchannel DNA sequencing matrices with a thermally controlled "viscosity switch".

Polymers and hydrogels that swell or shrink in response to environmental stimuli such as changes in temperature, pH, or ionic strength are of interest as switchable materials for applications in biotechnology. In this paper, we show that thermoresponsive polymers offer some particular advantages as entangled matrices for DNA sequencing by capillary and microchip electrophoresis. Matrices based on conventional water-soluble polymers demand a compromise in their design for microchannel electrophoresis: whereas highly entangled solutions of high molar mass polymers provide optimal sequencing performance, their highly viscous solutions require application of high pressures to be loaded into electrophoresis microchannels. Here, we demonstrate the reproducible synthesis, precise characterization, and excellent DNA sequencing performance of high molar mass, thermoresponsive polymer matrices that exhibit a reversible, temperature-controlled "viscosity switch" from high-viscosity solutions at 25 degrees C to low-viscosity, microphase-separated colloidal dispersions at a chosen, elevated temperature. The viscosity switch decouples matrix loading and sieving properties, enabling acceleration of microchannel flow by 3 orders of magnitude. DNA sequencing separations yielding read lengths of 463 bases of contiguous sequence in 78 min with 97% base-calling accuracy can be achieved in these matrices. Switchable matrices will be particularly applicable to microfluidic devices with dynamic temperature control, which are likely to provide the next major leap in the efficiency of high-throughput DNA analysis.

Base Sequence↗

PubMed2001

Impact of polymer hydrophobicity on the properties and performance of DNA sequencing matrices for capillary electrophoresis.

To elucidate the impact of matrix chemical and physical properties on DNA sequencing separations by capillary electrophoresis (CE), we have synthesized, characterized and tested a controlled set of different polymer formulations for this application. Homopolymers of acrylamide and N,N-dimethylacrylamide (DMA) and copolymers of DMA and N,N-diethylacrylamide (DEA) were synthesized by free radical polymerization and purified. Polymer molar mass distributions were characterized by tandem gel permeation chromatography - laser light scattering. Polymers with different chemical compositions and similar molar mass distributions were selected and employed at the same concentration so that the variables of comparison between them were hydrophobicity and average coil size in aqueous solution. We find that the low-shear viscosities of 7% w/v polymer solutions decrease by orders of magnitude with increasing polymer hydrophobicity, while hydrophilic polymers exhibit more pronounced reductions in viscosity with increased shear. The performance of the different matrices for DNA sequencing was compared with the same sample under identical CE conditions. The longest read length was produced with linear polyacrylamide (LPA) while linear poly-N,N-dimethylacrylamide (PDMA) gave approximately 100 fewer readable bases. Read lengths with DMA/DEA copolymers were lower, and decreased with increasing DEA content. This study highlights the importance of polymer hydrophilicity for high-performance DNA sequencing matrices, through the formation of robust, highly-entangled polymer networks and the minimization of hydrophobic interactions between polymers and fluorescently-labeled DNA molecules. However, the results also show that more hydrophobic matrices offer much lower viscosities, enabling easier microchannel loading at low applied pressures.

Acrylamides↗

PubMed2001

Attomole quantitation of protein separations with accelerator mass spectrometry.

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to subattomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5%. Micro-proton-induced X-ray emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phopsphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

Amino Acids↗

PubMed2001

The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis.

The ability of a polymer matrix to separate DNA by capillary electrophoresis (CE) is strongly dependent upon polymer physical properties. In particular, recent results have shown that DNA sequencing performance is very sensitive to both the average molar mass and the average coil radius of the separation matrix polymers, which are affected by both polymer structure and polymer-solvent affinity. Large polymers with high average molar mass provide the best DNA sequencing separations for CE, but are also the most challenging to characterize with accuracy. The methods most commonly used for the characterization of water-soluble polymers with application in microchannel electrophoresis have been gel permeation chromatography (GPC) and intrinsic viscosity measurements, but the limitations and potential inaccuracies of these approaches, particularly for large or novel polymers and copolymers, press the need for a more universally accurate method of polymer molar mass profiling for advanced DNA separation matrices. Here, we show that multi-angle laser light scattering (MALLS) measurements, carried out either alone or in tandem with prior on-line sample fractionation by GPC, can provide accurate molar mass and coil radius information for polymer samples that are useful for DNA sequencing by CE. Wider employment of MALLS for characterization of novel polymers designed as DNA separation matrices for microchannel electrophoresis should enable more rapid optimization of matrix properties and formulation, and assist in the development of novel classes of polymer matrices.

Electrophoresis, Capillary↗

PubMed2000

Isotope-labeled immunoassays without radiation waste.

The practice of immunoassay has experienced a widespread transition from radioisotopic labeling to nonisotopic labeling over the last two decades. Radioisotope labels have drawbacks that hamper their applications: (i) perceived radiation hazards of reagents, (ii) regulatory requirements and disposal problems of working with radioactive materials, and (iii) short shelf-life of the labeled reagents. The advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity, as is often the case with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay with the long-life isotope (14)C as the label and accelerator mass spectrometer (AMS) as the detection system. AMS quantifies attomole levels of several isotopes, including (14)C. With this exquisite sensitivity, the sensitivity of an immunoassay is limited by the K(d) of the antibody and not the detection system. The detection limit of the assays for atrazine and 2,3,7,8-tetrachlorodibenzo-p-dioxin was 2.0 x 10(-10) M and 2.0 x 10(-11) M, respectively, approximately an order of magnitude below the standard enzyme immunoassay. Notably, <1 dpm (0.45 pCi) of (14)C-labeled compound was used in each assay, which is well below the limit of disposal (50 nCi per g) as nonradioactive waste. Thus, endogenous reporter ligands quantified by AMS provide the advantages of an RIA without the associated problems of radioactive waste.

Animals↗

PubMed2000

Long-term kinetic study of beta-carotene, using accelerator mass spectrometry in an adult volunteer.

We present a sensitive tracer method, suitable for in vivo human research, that uses beta-[(14)C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 microgram 200 nCi) oral dose of beta-[(14)C]carotene was determined for 209 days in plasma. Analytes included beta-[(14)C]carotene, [(14)C]retinyl esters, [(14)C]retinol, and several [(14)C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of (14)C in plasma. Labeled beta-carotene and [(14)C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [(14)C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [(14)C]retinol in plasma was also delayed 5.5 h postdosing and its concentration rose linearly for 28 h before declining. Cumulative urine and stool were collected for 17 and 10 days, respectively, and 57.4% of the dose was recovered in the stool within 48 h postdosing. The stool was the major excretion route for the absorbed dose. The turnover times (1/k(el)) for beta-carotene and retinol were 58 and 302 days, respectively. Area under the curve analysis of the plasma response curves suggested a molar vitamin A value of 0.53 for beta-carotene, with a minimum of 62% of the absorbed beta-carotene being cleaved to vitamin A.In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of beta-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from beta-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for beta-carotene.

Adult↗

PubMed1999

Dose-dependent binding of ortho-phenylphenol to protein but not DNA in the urinary bladder of male F344 rats.

ortho-Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that is also known to be highly effective in inducing bladder tumors in male F344 rats. At present, neither the role of the urinary bladder in the bioactivation of OPP metabolites nor the nature of the molecular target is understood. To address these issues, we investigated the relationship between OPP dosage and macromolecular adduct formation in the urinary bladder of male F344 rats. Male F344 rats were treated with 0, 15, 50, 125, 250, 500, 1000 mg/kg of OPP and its radiocarbon analogue via oral gavage. The dosed rats were euthanized after 24 h, and the proteins were extracted from the liver, kidney, and bladder. The amount of radioactivity associated with the extracted protein was quantified using highly sensitive accelerator mass spectrometry. Protein binding in liver and kidney exhibited a linear or modest curvilinear relationship over the dose range studied. In the urinary bladder, however, a pronounced nonlinear relationship between protein adduct levels and administered dose was observed. The measured protein adduct levels were in agreement with the predicted concentrations of phenylbenzoquinone based on a proposed mechanism involving free phenylhydroquinone autoxidation in the urine. Unlike protein binding, DNA adducts measured from the same bladder samples did not show a significant difference from the control group. These data are consistent with the hypothesis that OPP is an indirect acting carcinogen, and that regenerative hyperplasia due to OPP-metabolite cytotoxicity and/or binding of OPP metabolites to protein targets may play an important role in OPP-induced bladder carcinogenesis.

Animals↗

PubMed1999

HPLC-accelerator MS measurement of atrazine metabolites in human urine after dermal exposure.

Metabolites of atrazine were measured in human urine after dermal exposure using HPLC to separate and identify metabolites and accelerator mass spectrometry (AMS) to quantify them. Ring-labeled [14C]atrazine was applied for 24 h with a dermal patch to human volunteers at low (0.167 mg, 6.45 muCi) and high (1.98 mg, 24.7 muCi) doses. Urine was collected for 7 days. The urine was centrifuged to remove solids, and the supernatant was measured by liquid scintillation counting prior to injection on the HPLC to ensure that < 0.17 Bq (4.5 pCi) was injected on the column. A reversed-phase gradient of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile became less polar with increasing time and separated the parent compound and major atrazine metabolites over 31 min on an octadecylsilane column. Peaks were identified by coelution with known standards. Elution fractions were collected in 1-min increments; half of each fraction was analyzed by AMS to obtain limits of quantitation of 14 amol. Mercapturate metabolites of atrazine and dealkylated atrazine dominated the early metabolic time points, accounting for approximately 90% of the 14C in the urine. No parent compound was detected. The excreted atrazine metabolites became more polar with increasing time, and an unidentified polar metabolite that was present in all samples became as prevalent as any of the known ring metabolites several days after the dose was delivered. Knowledge of metabolite dynamics is crucial to developing useful assays for monitoring atrazine exposure in agricultural workers.

Administration, Cutaneous↗

PubMed1999

Intrinsic erythrocyte labeling and attomole pharmacokinetic tracing of 14C-labeled folic acid with accelerator mass spectrometry.

Long-term physiologic tracing of nutrients, toxins, and drugs in healthy subjects is not possible using traditional decay counting of radioisotopes or stable isotope mass spectrometry due to radiation exposure and limited sensitivity, respectively. A physiologic dose of 14C-labeled folic acid (35 microg, 100 nCi) was ingested by a healthy adult male and followed for 202 days in plasma, erythrocytes, urine, and feces using accelerator mass spectrometry. All samples and generated wastes were classified nonradioactive and the subject received a lifetime-integrated radiological effective dose of only 11 microSv. Radiolabeled folate appeared in plasma 10 min after ingestion but did not appear in erythrocytes until 5 days later. Approximately 0.4% of the erythrocytes were intrinsically labeled with an average of 130 (14)C atoms during erythropoiesis from the pulse of plasma [14C]folate. An appropriate radiocarbon-labeled precursor can intrinsically label DNA or a specific protein during synthesis and obtain limits of quantitation several orders of magnitude below that of stable isotope methods.

Adult↗

PubMed1998

Analytical performance of accelerator mass spectrometry and liquid scintillation counting for detection of 14C-labeled atrazine metabolites in human urine.

Accelerator mass spectrometry (AMS) has been applied to the detection of 14C-labeled urinary metabolites of the triazine herbicide, atrazine, and the analytical performance of AMS has been directly compared to that of liquid scintillation counting (LSC). Ten human subjects were given a dermal dose of 14C-labeled atrazine over 24 h, and urine from the subjects was collected over a 7-day period. Concentrations of 14C in the samples have been determined by AMS and LSC and range from 1.8 fmol/mL to 4.3 pmol/mL. Data from these two methods have a correlation coefficient of 0.998 for a linear plot of the entire sample set. Accelerator mass spectrometry provides superior concentration (2.2 vs 27 fmol/mL) and mass (5.5 vs 54,000 amol) detection limits relative to those of LSC for these samples. The precision of the data provided by AMS for low-level samples is 1.7%, and the day-to-day reproducibility of the AMS measurements is 3.9%. Factors limiting AMS detection limits for these samples and ways in which these can be improved are examined.

Adult↗

PubMed1998

The dynamics of folic acid metabolism in an adult given a small tracer dose of 14C-folic acid.

Folate is an essential nutrient that is involved in many metabolic pathways, including amino acid interconversions and nucleotide (DNA) synthesis. In genetically susceptible individuals and populations, dysfunction of folate metabolism is associated with severe illness. Despite the importance of folate, major gaps exist in our quantitative understanding of folate metabolism in humans. The gaps exist because folate metabolism is complex, a suitable animal model that mimics human folate metabolism has not been identified, and suitable experimental protocols for in vivo studies in humans are not developed. In general, previous studies of folate metabolism have used large doses of high specific activity tritium and 14C-labeled folates in clinical patients. While stable isotopes such as deuterium and 13C-labeled folate are viewed as ethical alternatives to radiolabeled folates for studying metabolism, the lack of sensitive mass spectrometry methods to quantify them has impeded advancement of the field using this approach. In this chapter, we describe a new approach that uses a major analytical breakthrough, Accelerator Mass Spectrometry (AMS). Because AMS can detect attomole concentrations of 14C, small radioactive dosages (nCi) can be safely administered to humans and traced over long periods of time. The needed dosages are sufficiently small that the total radiation exposure is only a fraction of the natural annual background radiation of Americans, and the generated laboratory waste may legally be classified non-radioactive in many cases. The availability of AMS has permitted the longest (202 d) and most detailed study to date of folate metabolism in a healthy adult human volunteer. Here we demonstrate the feasibility of our approach and illustrate its potential by determining empirical kinetic values of folate metabolism. Our data indicate that the mean sojourn time for folate is in the range of 93 to 120 d. It took > or = 350 d for the absorbed portion of small bolus dose of 14C-folic acid to be eliminated completely from the body.

Area Under Curve↗

PubMedDate not supplied

Pyrethroid decrease in central nervous system from nerve agent pretreatment.

We studied the effect of pyridostigmine bromide, a nerve agent prophylactic, on the central nervous system (CNS) uptake of [14C]permethrin, a pyrethroid insecticide, at scaled human-equivalent exposures in rats using accelerator mass spectrometry (AMS). AMS detects 14C at attomole sensitivities and determines the tissue distribution of 14C-labeled compounds. Pyridostigmine bromide in chow at 7.75 mg kg(-1) per day lowered the CNS tissue levels of permethrin, dosed at 4.75 microg kg(-1), in the CNS of rats by 30%. These results are inconsistent with hypothesized synergy of such compounds as a precursor to 'Gulf War syndrome'.

Animals↗