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PubMed2006

Microanalyzer for biomonitoring lead (Pb) in blood and urine.

Biomonitoring of lead (Pb) in blood and urine enables quantitative evaluation of human occupational and environmental exposures to Pb. State-of-the-art ICP-MS instruments can only analyze metals in laboratories, resulting in lengthy turnaround times, and they are expensive. In response to the growing need for a metal analyzer capable of on-site, real-time monitoring of trace toxic metals in individuals, we developed a portable microanalyzer based on flow-injection/stripping voltammetry (ASV), and validated the system using rat blood and urine spiked with known amounts of Pb. Fouling of electrodes by proteins often prevents the effective use of electrochemical sensors in biological matrices. Minimization of such fouling was accomplished with suitable sample pretreatment and by establishing turbulent flow of blood and urine containing Pb onto the electrode inside the microanalyzer, which resulted in no apparent electrode fouling even when the samples contained 50% urine or 10% blood by volume. No matrix effect was observed for the voltammetric Pb signals, even when the samples contained 10% blood or 10% urine. The microanalyzer offered linear concentration ranges relevant to Pb exposure levels in humans (0-20 ppb in 10% blood samples, 0-50 ppb in 50% urine samples). The device showed excellent sensitivity and reproducibility; Pb detection limits were 0.44 ppb and 0.46 ppb, and % R.S.D. was 4.9 and 2.4 in 50% urine and 10% blood samples, respectively. It gave similar Pb concentrations in blood and urine to those measured by ICP-MS. It offered high throughput (3 min per sample) and economical use of samples (60 microL per measurement) as well as low reagent consumption (1 microg of Hg per measurement), thus minimizing environmental concerns associated with mercury use. Since it is miniaturized, the microanalyzer is portable and field-deployable. Thus, it shows much promise as the next-generation analyzer for the biomonitoring of toxic metals.

Animals↗
PubMed2001

Validated method for the determination of the novel organo-ruthenium anticancer drug NAMI-A in human biological fluids by Zeeman atomic absorption spectrometry.

NAMI-A is a novel ruthenium-containing experimental anticancer agent. We have developed and validated a rapid and sensitive analytical method to determine NAMI-A in human plasma, plasma ultrafiltrate and urine using atomic absorption spectrometry with Zeeman correction. The sample pretreatment procedure is straightforward, involving only dilution with an appropriate hydrochloric acid buffer-solution. Because the response signal of the spectrometer depended on the composition of the sample matrix, in particular on the amount of human plasma in the sample, all unknown samples were diluted to match the matrix composition in which the standard line was prepared (plasma-buffer 1:10 v/v). This procedure enabled the measurement of samples of different biological matrices in a single run. The validated range of determination was 1.1-220 microM NAMI-A for plasma and urine, and 0.22-44 microM for plasma ultrafiltrate. The lower limit of detection was 0.85 microM in plasma and urine and 0.17 microM in plasma ultrafiltrate. The lower limit of quantitation was 1.1 and 0.22 microM, respectively. The performance of the method, in terms of precision and accuracy was according to the generally accepted criteria for validation of analytical methodologies. The applicability of the method was demonstrated in a patient who was treated in a pharmacokinetic phase I trial with intravenous NAMI-A.

Antineoplastic Agents↗
PubMed2005

beta-Catenin and met deregulation in childhood Hepatoblastomas.

Activation of the Wnt/beta-catenin and hepatocyte growth factor/Met signaling has been implicated in various tumors. Owing to the cross-talk between these pathways and aberrant redistribution of beta-catenin in hepatoblastomas, we examined their status in this tumor. This study examined changes in beta-catenin and Met in paired pretreatment and post-treatment hepatoblastoma tissues in relation to their effects on proliferation and target genes such as c-myc and cyclin-D1. In this study we compared proliferation indices, beta-catenin staining and its known molecular targets, c-myc and cyclin-D1, and Met, a tyrosine kinase receptor for hepatocyte growth factor in pretreatment and post-treatment specimens. Pretreatment and post-treatment sections from 13 children, ages 11 weeks to 9 years, were analyzed for these markers by immunohistochemistry. All tumors (13 of 13) displayed increased proliferation and beta-catenin (cytoplasmic and nuclear) staining in pretreatment biopsies that remained relatively unaffected after treatment. Aberrant Met staining (cytoplasmic) was observed in all pretreatment samples that decreased considerably after treatment in 11 of 13 patients. A significant subset of these tumors showed increased c-myc and cyclin-D1 staining in pretreatment biopsies that decreased after chemotherapy in most cases. beta-Catenin redistribution in tumor cells corresponds to proliferation in hepatoblastomas. However, beta-catenin nuclear localization remains unaffected in viable hepatoblastoma tissue after chemotherapy. In contrast, Met undergoes a prominent decrease after treatment and thus might be important in pathogenesis of hepatoblastoma.

Biomarkers, Tumor↗
PubMed2001

[Determination of anions in liquid crystal samples by UV-photolysis-ion chromatography].

A new method for the determination of anions in liquid crystal samples by UV-photolysis-ion chromatography was developed. The sample pretreatment method, the photolytic conditions and the chromatographic parameters were optimized. The results indicate that there have five anions (F-, Cl-, NO2-, Br-, I-) in the liquid crystal sample. By the filtration with the Dionex OnGuard RP and P cartridges, most of the organic compounds in the photolyzed sample solution can be removed. In the process of photolysis, the efficiency can be remarkably enhanced by the adding of H2O2. Iodide in the liquid crystal sample can be accurately determined by adding of 0.1 mmol/L NaOH. In the five anions, fluoride, choloride, nitrite and bromide can get their maximal responses when the photolysis time is 2 h. With the strongest reducibility, iodide only needs 1.5 h to get the highest response. The separation of the five anions was accomplished with Dionex IonPac AS16 column and the determination can be completed in 25 min. The recoveries for the five anions were between 85.1% and 111.5%.

English Abstract↗
PubMed1994

A direct membrane filter method for enumerating somatic coliphages in drinking water.

The application of a simple membrane filter method to enumerate specific somatic bacteriophages of Escherichia coli, using E. coli C as host strain, from drinking water samples was studied. The efficiency of the method using cellulosic membrane filters, samples pretreated with magnesium ions and Tween 80 added to agar medium-host cell lawns ranged from 68.9 to over 112%, depending on the phage content of the sample. To avoid the pre-treatment of the sample with magnesium salts, electropositive-charged filters of cellulosic ester (HA-PEI and HA-Nalco) and Virosorb-1MDS filters were tested in conjunction with the simple membrane filter method. The electropositive filters showed wide bacteriophage recovery rate intervals depending on the sample treatment, ranging between 31.4 and 96.2% for ester-type filters, and a mean recovery lower than 2.2% for Virosorb filters. On the other hand, it was proved that the use of Tween 80 as an eluent improved somatic coliphage recovery rates for all the filters tested. In short, this methodology provides a rapid analysis (6-8 h) of the somatic coliphages from drinking water using the membrane filtration technique.

Adsorption↗
PubMed2005

Analysis of tetrabromobisphenol A and other phenolic compounds in water samples by non-aqueous capillary electrophoresis coupled to photodiode array ultraviolet detection.

Non-aqueous capillary electrophoresis (NACE) with large-volume sample stacking injection using the electroosmotic flow pump (LVSEP) has been developed for the determination of tetrabromobisphenol A (TBBPA) and other phenolic compounds in environmental matrices. Methanol has been used as run buffer solvent to reduce the electroosmotic flow (EOF). Identification and quantification of the analytes was performed by photodiode array ultraviolet detection. LVSEP-NACE improved sensitivity of the peak height by 90-300-fold. The method developed was applied to the analysis of TBBPA in river water and wastewater samples, using solid-phase extraction (SPE) as sample pretreatment process. The average recoveries of the analytes were in the range of 96-106% and 73-103% for 1 L of river water and 0.5 L of wastewater samples, respectively. When the method was based on off line SPE-LVSEP-NACE, sensitivity was improved by 3300-4500-fold and 1600-2200-fold for river water and wastewater samples, respectively.

Electrophoresis, Capillary↗
PubMed1984

Automated high-pressure liquid chromatographic assay for antiepileptic drugs and their major metabolites by direct injection of serum samples.

We present a fully automated high-pressure liquid chromatographic (HPLC) assay for simultaneous quantitation of five antiepileptic drugs [ethosuximide (ES), primidone, phenobarbital, phenytoin, and carbamazepine] and their major metabolites [phenylethylmalondiamide (PEMA), carbamazepine-10,11-dihydro-10,11-diol (CBZD), and carbamazepine-10,11-epoxide (CBZE)] in serum samples without sample pretreatment. Two other metabolites, p-hydroxyphenobarbital (HPB) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), can be separated as well. Interference with serum constituents prevents a quantitative determination. Serum samples are injected onto a pre-column, the biological matrix is eluted with water, and the compounds of interest are subsequently separated on an analytical column. The anticonvulsants are eluted at 60 degrees C with a mobile phase containing acetonitrile:water (21:80 by vol) at a flow rate of 1.0-2.0 ml/min (flow-step gradient). The eluted compounds are monitored at 200 nm. Each analysis requires about 30 min. Analytical recoveries varied from 90 to 98%. Within-day coefficients of variation (CVs) ranged from 0.9 to 4.9%, between-day CVs from 2.8 to 7.6%. The lower limit of detection for all drugs is 0.2 microgram/ml serum, with a sample size of 100 microliters. At therapeutic concentrations we achieved baseline separation for all eight compounds, and there was no interference from other drugs.

Acetonitriles↗
PubMedDate not supplied

Reparative response of human articular cartilage in tissue culture. Comparison between a normal and an osteoarthritic knee of the same donor.

Some evidence is presented that the metabolism of visually intact cartilage taken from a knee with focal osteoarthritic lesions, may already be affected. Proteoglycan metabolism of articular cartilage from the macroscopically unaffected right knee and the osteoarthritic left knee of the same donor was assayed in long-term tissue culture. A good reparative response was found in the right knee with an increase in the relative amount of radiolabelled proteoglycan and proteoglycan aggregates, and in the total amount of labelled proteoglycan per mg dry tissue. No significant differences were found when the anteromedial, anterolateral and posterolateral areas were compared. Visually intact samples taken from the left knee, with a deep osteoarthritic cartilage erosion on the medial condyle, showed no accumulation of radiolabel into proteoglycan and proteoglycan aggregates for the enzymatically pretreated samples. This was interpreted as a repair failure.

Cartilage, Articular↗
PubMed2006

Capillary electrophoresis of proteins 2003-2005.

This review article with 304 references describes recent developments in CE of proteins, and covers the two years since the previous review (Hutterer, K., Dolník, V., Electrophoresis 2003, 24, 3998-4012) through Spring 2005. It covers topics related to CE of proteins, including modeling of the electrophoretic migration of proteins, sample pretreatment, wall coatings, improving separation, various forms of detection, special electrophoretic techniques such as affinity CE, CIEF, and applications of CE to the analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals, and recombinant protein preparations.

Animals↗
PubMed2001

Measurement of nephrolithiasis urinary markers by capillary electrophoresis.

A previously developed method for screening organic acidurias by capillary electrophoresis has been validated for oxalate and citrate measurement in urine. Sample pretreatment is minimum, just acidification and centrifugation. Detection is by direct UV. Validation parameters of the method can be considered adequate. Response is linear for both analytes in standards and samples. The assayed ranges were 200-1,000 mg/l for citrate and 10-200 mg/l for oxalate. Recoveries ranged from 99.4+/-3 to 101.7+/-2.4%, maximum imprecision in oxalate concentration was of 7.6% RSD and limits of detection in samples were 0.67 mg/l for oxalate and 25.9 mg/l for citrate, both lower than the measured values in samples. Identification of increased glyoxylic (oxoacetic acid) and glyceric acids (2,3-dihydroxy propanoic) are also included to facilitate the diagnosis.

Biomarkers↗
PubMed2004

Oxygen, carbon, and sulfur segregation in annealed and unannealed zerovalent iron substrates.

Finely ground and pretreated iron substrates known as "zerovalent iron" or "Fe0" are used as reductants in the environmental remediation of halogenated hydrocarbons, and the composition of their surfaces significantly affects their reactivity. Samples of unannealed and annealed (heat-treated under H2/N2) zerovalent iron were analyzed using X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES). Surface concentration of the iron and of the impurities observed by XPS and AES, carbon, chlorine, sulfur, and oxygen, were measured before and after soaking in trichloroethylene (TCE) and in water saturated with TCE (H2O/TCE) to simulate chlorocarbon remediation conditions. Samples pretreated by annealing at high temperature under H2 contained less iron carbide. The carbide contaminant was evident in both iron and carbon XPS spectra, with binding energies of 709.0 and 283.3 eV for the Fe 2p3/2 and C 1s, respectively. The annealed Fe0 surface also contained more sulfur. The carbide concentration was essentially unchanged by TCE and H2O/TCE exposure, whereas the sulfur decreased in proportion to chlorine adsorption following the dechlorination reaction. While oxygen concentration is initially lower on the annealed substrate surface, it rapidly increased during the model TCE remediative treatment process and thus does not represent a significant effect of the annealing process on surface reactivity.

Journal Article↗
PubMed2004

Comparison of two field sampling procedures (En Core and field methanol extraction) for volatile organic compounds.

In-situ Lasagna technology was recently evaluated at a contaminated site at Offutt Air Force Base. The site was contaminated with low levels (<30 mg/kg) of volatile organic compounds (VOCs). Originally, researchers planned to use field methanol extraction for both pre- and post-treatment sampling to evaluate the effectiveness of the technology on contaminant reduction. Precharacterization sampling, however, indicated that concentrations of some contaminants of concern were much lower than expected. Because use of methanol increases the detection limit, it was probable that post-treatment concentrations of these target contaminants would be nondetectable if methanol extraction was used. Project management, therefore, decided to use En Core samplers in addition to field methanol extraction during the pretreatment sampling event. The En Core sampling approach, while yielding a lower detection limit, uses discreet samples along the length of a core, whereas the methanol extraction approach samples the entire length of the core. The concern was that discreet samples may bias results if any "hot spots" were present. The two field sampling procedures, En Core and field methanol extraction, were performed side-by-side during the pretreatment phase of the technology evaluation in order to determine if the concern was valid for this site. Results were compared for four contaminants of concern: trichloroethylene, vinyl chloride, cis-1,2-dichloroethene, and trans-1,2-dichloroethene. The two procedures produced similar results with respect to both the concentration means and the variances, and no bias was evident. This finding supports project management's decision to use only En Core samplers post-treatment due to low concentrations of target contaminants.

Environmental Monitoring↗
PubMed2005

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone.

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.

Acrosome↗
PubMed1994

Measurement of S-phase fraction and ploidy in sequential fine-needle aspirates from primary human breast tumours treated with tamoxifen.

Sequential fine-needle aspirates (FNAs) for cytodiagnosis and flow cytometry were taken from 21 patients with primary breast carcinoma at intervals ranging from 1 to 3 months after the commencement of first-line tamoxifen therapy. Nine patients achieved a sustained complete or near complete response over a 3-9 month period. The tumour cells from seven out of nine of these patients were initially aneuploid, while the remaining two patients had diploid tumours. An analysis of sequential FNAs showed that, in three out of the seven aneuploid tumours, only benign epithelial cells could be detected by cytology in the post-tamoxifen sample. In the remaining six cases, including the two diploid tumours, there was no change in ploidy but a reduction in S-phase fraction (SPF) to approximately 50% of the pretreatment level. In all cases, these changes in ploidy or SPF were seen with a mean lead time of 4 months before the tumour had reached clinical complete remission. None of these patients have relapsed after a mean follow-up period of 18 months. The tumours of 12 patients achieved no more than a temporary partial response to primary tamoxifen therapy. In seven out of eight of these cases, which were all initially aneuploid, sequential FNAs during tamoxifen therapy revealed either an increase or no change in the SPF with the tumour remaining aneuploid. In the remaining four cases the tumours were all recorded as being diploid in the pretreatment sample. However, although three of these cases had a temporary partial response to tamoxifen, an aneuploid component was picked up in repeat sequential FNAs with a mean lead time of 5 months before clinical confirmation of eventual disease progression. We conclude that changes in ploidy and SPF detected by flow cytometry may predict initial response and the likelihood of relapse of breast tumours to tamoxifen before clinical changes become evident. These data justify a larger study.

Biomarkers↗
PubMed1965

STATISTICAL ANALYSIS OF MOLLUSCICIDE FIELD TRIALS.

The paper describes the methods used in the statistical analysis of field trials of acrolein, Bayluscide, ICI 24223 and NaPCP molluscicides. The study shows the importance of adequate sampling of the snail population both before and after the application of the molluscicide and the necessity of obtaining equally complete data from the controls. For kills above 99%, if the pretreatment collection contains fewer than 400-500 snails, the range between the confidence limits exceeds 5% and, if the pretreatment sample contains fewer than 1200-1500 snails, the range exceeds 2%. For all the molluscicides except acrolein, a cube-root transformation was found to be applicable. After treatment with acrolein, the snails were distributed at random. It most of the trials there were significant changes in the snail populations in the control canals, possibly due to sampling. No significant differences were observed between samplings taken 48 hours and one week after treatment; one of these could be omitted in future trials.

Africa↗
PubMed1994

The sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls.

Scrotal insulation and dexamethasone treatment were used as a model to compare the effect of testicular heating and stress on spermatogenesis. Insulation was applied to the scrotum of eight bulls (insulated) for a period of four days, eight bulls were treated daily for seven days with 20 mg dexamethasone injected intramuscularly, and four bulls were untreated controls. Semen from four bulls in each group was collected and evaluated over a six-week period after treatment. Blood samples for testosterone analysis were taken hourly for eight hours at the beginning and the end of the six-week period from the control bulls and before and after treatment from the four insulated and four dexamethasone-treated bulls that were not used for semen collection. At the end of the last blood sampling period, the four bulls in each group were castrated for the collection of testicular tissue for the determination of testosterone concentrations. Basal, peak episodic, and mean serum testosterone concentrations among control bulls, pre and postinsulated bulls, and pretreatment samples of dexamethasone-treated bulls were not different (p > 0.05); however, bulls that had received dexamethasone treatments had significantly lower basal, peak episodic, and mean testosterone concentrations (p < 0.05). Tissue concentrations of testosterone in control, insulated, and dexamethasone-treated bulls were not significantly different but tended to be lower in dexamethasone-treated bulls (p > 0.13). The spermiograms of the control bulls varied insignificantly over the six-week sampling period; however, there was a marked increase in sperm defects in insulated and dexamethasone-treated bulls. The types of sperm defects and the temporal relationships of rises and declines of sperm defects were quite similar for both treatments. All bulls recovered to approximately pretreatment levels of sperm defects by six weeks after the initiation of treatment. Results indicate that two of the most common types of insults to spermatogenesis in bulls, heat and stress, result in similar spermiograms.

Animals↗
PubMed2003

Capillary electrophoresis for short-chain organic acids and inorganic anions in different samples.

This review article is a comprehensive survey of capillary electrophoresis methods developed for the measurement of short-chain organic acids and inorganic anions in a wide variety of matrices, such as food and beverages, environmental, industry, and other applications, as well as clinical applications in body fluids such as urine, plasma or cerebrospinal fluid. Details of sample pretreatment and of electrophoretic conditions have been collected in tables, arranged by the type of matrix. Strategies employed for method development for the analysis of these compounds by capillary electrophoresis in real samples are discussed.

Acids↗
PubMed2003

Isotope-dilution ICP-MS for trace element determination and speciation: from a reference method to a routine method?

This critical review discusses the conditions under which inductively coupled plasma-isotope dilution mass spectrometry (ICP-IDMS) is suitable as a routine method for trace element and element-speciation analysis. It can, in general, be concluded that ICP-IDMS has high potential for routine analysis of trace elements if the accuracy of results is of predominant analytical importance. Hyphenated techniques with ICP-IDMS suffer both from lack of commercially available isotope-labeled spike compounds for species-specific isotope dilution and from the more complicated system set-up required for species-unspecific ICP-IDMS analysis. Coupling of gas or liquid chromatography with species-specific ICP-IDMS, however, enables validation of analytical methods involving species transformations which cannot easily be performed by other methods. The potential and limitations of ICP-IDMS are demonstrated by recently published results and by some unpublished investigations by our group. It has been shown that possible loss of silicon as volatile SiF4 during decomposition of a sample by use of hydrofluoric acid has no effect on trace silicon determination if the isotope-dilution step occurs during digestion in a closed system. For powder samples, laser ablation ICP-IDMS can be applied with an accuracy comparable with that only available from matrix-matched standardization, whereas the accuracy of electrothermal vaporization ICP-IDMS was strongly dependent on the element determined. The significance of easy synthesis of isotope-labeled spike compounds for species-specific ICP-IDMS is demonstrated for monomethylmercury and Cr(VI). Isotope-exchange reactions between different element species can prevent the successful application of ICP-IDMS, as is shown for iodinated hydrocarbons. It is also shown for monomethylmercury that species transformations during sample-pretreatment steps can be followed by species-specific ICP-IDMS without loss of accuracy. A relatively simple and time-efficient procedure for determination of monomethylmercury in environmental and biological samples is discussed. The method, which entails a rapid microwave-assisted isotope dilution step and in-situ extraction of the derivatized species, has good potential for routine application in the future.

Journal Article↗