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PubMed1991

An in vivo study on the release of fluoride from glass-ionomer cement.

Fluoride release from a glass-ionomer cement was measured in vivo during an 8-day period. Maxillary acrylic resin appliances, each carrying four glass-ionomer cement specimens, were worn by four subjects at night. Unstimulated whole saliva samples were collected before the subjects retired to bed (pretreatment samples) and in the morning (posttreatment samples). The average fluoride concentration and saliva flow of pretreatment samples were 31 +/- 4 ng/mL and 0.5 +/- 0.1 mL/min, respectively. In all subjects there was a statistically significant posttreatment increase (.005 less than P less than .025) in salivary fluoride concentration. The release of fluoride was nearly constant during the test period.

Adult↗

PubMed1996

Prolongation of survival of alloskin grafts with no concurrent general suppression of the burned patient's immune system: a preliminary clinical investigation.

The adequate management of burns is the requisite to save the patient's life, to prevent the onset of secondary infections and to obtain healing with acceptable cosmetic results. Previous data have shown that the use of alloskin grafts in the acute phase of burns yields the best clinical results. Unfortunately, this approach is curbed by the patient's immune response which induces the rejection of the allografts. In this report we endeavoured to solve this problem by ways that would not imply the general suppression of the patient's immune system. Thus, we can now report that a longer survival of alloskin grafts can be induced in situ by pretreating the alloskin samples to be grafted with both an anti-beta 2-microglobulin monoclonal antibody (beta 2mAb) and irradiation with ultraviolet-C light (UVC). The advantage of our novel approach is that it does not need any concomitant administration of immunosuppressive agent(s) to the burned patients. In this study, we followed five severely burned patients grafted with alloskin samples pretreated with either beta 2mAb or beta 2mAb plus UVC irradiation. In comparison with untreated alloskin samples from the same source grafted in parallel, the mean survival time (MST) of the beta 2mAb-pretreated alloskin specimens increased by 35 per cent (i.e. from 18.3 +/- 3.2 days to 24.7 +/- 5.5 days) in three patients. Moreover, the MST of the alloskin grafts pretreated with both beta 2mAb and UVC irradiation was lengthened by 100 per cent (i.e. from 18.5 +/- 4.5 days to 37.0 +/- 8.0 days) in the remaining two patients. These preliminary results lend credence to the view that local immune suppression could be achieved in situ by our approach via: (1) the impairment of the proper functions of the HLA-class I antigen by the bound beta 2mAb; and (2) the inhibition of immune reactions taking place inside the alloskin grafts by some immunosuppressive signal(s) generated by the UVC-pretreated alloepidermal cells. Hence, our approach stands as an exciting and useful alternative to the otherwise well-known, deleterious general suppression of the burned patient's immune system.

Adolescent↗

PubMed1986

Single radial immunodiffusion of serum apolipoproteins C-II, C-III and E--pretreatment of samples with surfactant.

An assay of apolipoproteins (Apo) C-II, C-III and E in human sera by single radial immunodiffusion (SRID) is described. The procedure involves pretreatment of the serum sample with surfactant to facilitate diffusion of very low density lipoproteins in agarose. This allowed measurement of apolipoproteins in lipidemic sera by SRID in agarose gel. The intraplate coefficient of variation was less than 2.5%, and the interplate coefficient of variation, less than 3.0%. Correlation with double-antibody radioimmunoassay produced the following coefficients of correlation: gamma = 0.9931 (n = 17) for Apo C-II, gamma = 0.9221 (n = 33) for Apo C-III, and gamma = 0.9340 (n = 17) for Apo E.

Apolipoprotein C-II↗

PubMed1991

Methods compared for determining activity of N-acetyl-beta-D-glucosaminidase in urine without pretreatment of sample: different sensitivity and species effect.

N-Acetyl-beta-D-glucosaminidase (NAG) activities in the urine of men and rats were measured with methods recommended as procedures without pretreatment of the urine sample. Four different derivatives of NAG were compared for determination: 4-nitrophenyl; 3,3-dichlorophenylsulfonphthaleinyl; 3-cresolsulfonphthaleinyl, and 2-methoxy-4-(2-nitro-vinyl)phenyl. The conventional test using the 4-nitrophenyl derivative showed the highest activities and correlated very well with the other tests. There are method-dependent differences between NAG activities measured in men and rats due to the different Km values and inhibitory effects by urea.

Acetylglucosamine↗

PubMed1996

Automation of selective assays for on-line bioprocess monitoring by flow-injection analysis.

On-line analysis of one component in a complex media used for bioprocesses requires the application of selective tests such as enzymes assays. Because these assays are susceptible to interference by other medium components and have a limited detection range, automatic sample pretreatment is a prerequisite. The progress made with automatic sample pretreatment in flow-injection analysis makes this technique particularly suitable for on-line monitoring of bioprocesses. Moreover, newly developed software control systems may improve the necessary robustness of flow-infection analysis systems.

Animals↗

PubMed1994

[An improved method for determination of C-peptide levels in serum: pretreatment of test samples with anti-insulin antibody insolubilized with magnetic particles].

RIA using anti-C-peptide antibody has been employed for determination of C-peptide in serum. However, the level by this method is a sum of the levels of C-peptide and proinsulin, because anti-C-peptide cross-reacts with proinsulin. This time we developed an improved assay for C-peptide, in which a test sample is pretreated with anti-insulin antibody to eliminate proinsulin in samples before C-peptide assay. The assay procedure is composed of 3 steps; the first is the incubation of test serum with anti-insulin antibody insolubilized with magnetic particles to form a complex of proinsulin-anti-insulin antibody, the 2nd step is the centrifugation of the mixture to eliminate the complex and the last step is the assay for C-peptide by use of RIA kit in the supernatant. The assay is simple, sensitive and reproducible. Serum C-peptide level by this method is not influenced by the presence of proinsulin in test serum, even when as high as 9 ng/ml of proinsulin is contained. This assay revealed that a patient with insulinoma had normal level of serum C-peptide in spite of the extremely high level of proinsulin.

C-Peptide↗

PubMedDate not supplied

Fecal polymerase chain reaction with 16S ribosomal RNA primers can detect the presence of gastrointestinal Helicobacter in dogs.

Questions about pathogenesis and therapy for Helicobacter infections in dogs could be answered with a simple, noninvasive, sensitive, and specific diagnostic test. We hypothesized that a fecal polymerase chain reaction (PCR) assay would detect Helicobacter and could be useful for assessing therapeutic responses. Paired gastric biopsies and fecal samples were obtained from 39 random source dogs (group 1). Gastric biopsies from each of these dogs had histologic evidence of gastric spiral bacteria, and paired gastric tissue and fecal samples produced a 375-base pair (bp) product when amplified by PCR with Helicobacter-specific primers. Specificity of the PCR product was confirmed by detection of expected 60-, 119-, and 196-bp products following Hinfl digestion. Direct sequencing of amplicons from paired PCR products from gastric biopsy and fecal samples from 8 group I dogs showed that gastric products had the highest homologies with known gastric Helicobacter species, whereas fecal products had the highest homologies with intestinal species. Healthy mixed-breed dogs (group II; n = 8) with histologically confirmed spiral bacteria infection were treated with a 21-day course of metronidazole, amoxicillin, and famotidine. Fecal samples were collected from group II dogs twice before and within 3 days of completion of treatment. The PCR results correctly identified 15/16 pretreatment samples as positive: 1 pretreatment sample was negative. PCR results identified 8/8 posttreatment samples as Helicobacter negative. Fecal PCR is a useful test for detecting Helicobacter infection in dogs. This assay may be useful as a screening test for infection and could be used to address questions relevant to pathogenesis and therapy.

Animals↗

PubMed1995

Determination of paclitaxel and metabolites in mouse plasma, tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography.

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6 alpha, 3'-p-dihydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6 alpha-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1-0.2 g/ml) in bovine serum albumin (40 g/l) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200-1000 microliters of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut columns. 2'-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 to 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 microliters of sample. In tissues, faeces and urine the LLQs are 25-100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 microliters (faeces: 200 microliters) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25-100,000 ng/ml for paclitaxel and 25-500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.

Animals↗

PubMed2005

[Progress in the determination of trace polycyclic aromatic hydrocarbons in complex matrices].

The analytical methods of polycyclic aromatic hydrocarbons (PAHs) in different samples, e.g., vegetable oils, waters and soils, have been reviewed. The sample pretreatment steps play a key role during the analysis due to the very low PAHs contents. The pretreatment methods for water samples include liquid-liquid extraction, solid phase extraction, etc. The pretreatment methods for soil samples include Soxhlet extraction, ultrasonic extraction, microwave assisted extraction, accelerated solvent extraction, and supercritical fluid extraction, etc. The pretreatment methods for vegetable oils include saponification, liquid-liquid partitioning, caffeine complexation, solid phase extraction, and supercritical fluid extraction, etc. Gas chromatography with mass spectrometry and high performance liquid chromatography (HPLC) with fluorescence detection are mainly used for separation and detection. The novel on-line determination method, e.g., the coupling of liquid chromatography with high performance liquid chromatography, or with gas chromatography may obtain ideal results. Fifty-two references are cited.

Chromatography, Gas↗

PubMed2004

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids.

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.

Automation↗

PubMed1990

High-performance liquid chromatographic bio-analysis and preliminary pharmacokinetics of the experimental antitumour drug vintriptol.

A high-performance liquid chromatographic procedure, including sample pretreatment, is presented for the analysis of the experimental antitumour drug vintriptol in plasma. The sample pretreatment involved liquid-liquid extraction of the buffered (pH 3) sample with chloroform. Vinblastine was used as internal standard. Separation was achieved on a Hypersil ODS (5 microns) column with a mobile phase of acetonitrile-phosphate buffer. Electrochemical detection (at +0.70 V) was used, giving a detection limit of 2 micrograms/l. The applicability of the assay was demonstrated in a pharmacokinetic study with eight cancer patients who received 45 or 50 mg/m2 vintriptol in a phase I study. A three-compartment model was used to fit the plasma concentration-time curves. Pharmacokinetic parameters are presented.

Antineoplastic Agents↗

PubMed1996

Direct determination of serum glycyrrhetic acid by a monoclonal antibody-based inhibition ELISA using ibuprofen for releasing serum albumin-bound glycyrrhetic acid.

We found that ibuprofen (IBU) had a potential for releasing serum albumin-bound glycyrrhetic acid (GA). Based on this observation, IBU was used to pretreat samples for the determination of serum GA levels by an inhibition ELISA. This method, termed IBU method was evaluated by the recovery of GA from human serum albumin (HSA) or normal human serum (NHS) that contained the exogeneously added GA (37-1000 ng/ml). The mean recovery of GA from HSA and NHS samples treated with IBU were 104.7 and 105.2%, respectively, whereas those without IBU pretreatment were 2.8 and 10.7%, respectively. Comparison of IBU method and chloroform extraction method revealed that the GA content of serum samples pretreated by each method were almost the same. These results suggest that IBU method is useful as a serum processing procedure for the determination of serum GA levels by an inhibition ELISA.

Antibodies, Monoclonal↗

PubMed1997

[Design and application of vacuum manifold for solid phase extraction].

Solid phase extraction (SPE) is now a common method for sample pretreatment. SPE vacuum manifold is a key instrument when SPE is used for sample pretreatment. It was developed by National Chromatographic R. & A. Center. Being connected with a vacuum pump, it can deal with 12 samples simultaneously. The manifold has a unique rotatable cover and can be conveniently rotated when switching between any two steps of SPE. An application of this SPE vacuum manifold in measuring bile acids in human serum was given.

Bile Acids and Salts↗

PubMed1997

Selection and transplantation of autologous hematopoietic CD34+ cells for patients with multiple myeloma.

Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

Antigens, CD34↗

PubMed1994

In vivo rodent erythrocyte micronucleus assay.

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (474) for easy reference. Introduction, purpose, scope, relevance, application and limits of test. The analysis of immature erythrocytes in either bone marrow or peripheral blood is equally acceptable for those species in which the spleen does not remove micronucleated erythrocytes. In the mouse, mature erythrocytes are also an acceptable cell population for micronucleus analysis when the exposure duration exceeds 4 weeks. Test substances. Organic solvents such as DMSO are not recommended. Freshly prepared solutions or suspensions should be used unless stability data demonstrate the acceptability of storage. Vegetable oils are acceptable as solvents or vehicles. Suspension of the test chemicals is acceptable for p.o. or i.p. administration but not for i.v. injection. The use of any unusual solvent should be justified. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and square root of 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2000

Direct high-performance liquid chromatographic determination of (R)- and (S)-propranolol in rat microdialysate using on-line column switching procedures.

Two different column-switching HPLC systems (CSWs), employing restricted access material for initial pretreatment of biological samples, were developed for the determination of propranolol enantiomers in microdialysate. CSW 1 was a single-pump set-up based on an initial sample clean-up step with a RP-18 ADS precolumn coupled with an ovomucoid analytical column for direct drug enantioseparation. For the two-pump column set-up (CSW 2), a teicoplanin analytical column was applied for the enantioselective assay after initial sample pretreatment using a RP-8 ADS precolumn. The inter-day precision of the CSW 1 ranged from 0.5 to 5.1% for (R)-propranolol and from 5.1 to 10.5% for (S)-propranolol. The limit of detection (LOD) was set at 10 ng/ml and 15 ng/ml for (R)- and (S)-propranolol, respectively. Inter-day relative standard deviation values of the CSW 2 ranged from 1.1 to 9.9% for (R)-propranolol and from 1.3 to 9.6% for (S)-propranolol. The LOD of the method was 3.0 ng/ml for (R)-propranolol and 2.5 ng/ml for (S)-propranolol. Both approaches were successfully applied for stereoselective monitoring of unbound propranolol levels in rat microdialysates.

Adrenergic beta-Antagonists↗

PubMedDate not supplied

[Application of high-pressure liquid chromatography in the analysis of urinary metabolites of aromatic solvents].

The introduction of HPLC methods in industrial toxicology represents a valuable tool for the routine monitoring of workers occupationally exposed to aromatic solvents. The HPLC method described here permits the simultaneous determination of metabolites of ethylbenzene, styrene, toluene, xylene isomers, benzene, phenol and cresol isomers in diluted urine samples. Pretreatment of urine samples with steam distillation is necessary only for determination of phenol and cresols because of their low concentrations. A comparison between a GLC and the HPLC procedure for mandelic and phenylglyoxylic acids confirmed the satisfactory performance of the HPLC method.

Adult↗

PubMed1996

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells.

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.

Adult↗