In vivo monitoring of multiple trace metals in the brain extracellular fluid of anesthetized rats by microdialysis-membrane desalter-ICPMS.

We have developed an on-line analytical system involving microdialysis (MD) sampling, a carbohydrate membrane desalter (CMD), and an inductively coupled plasma mass spectrometer (ICPMS) system for the simultaneous determination of multiple trace metals in the extracellular fluid (ECF) in the brains of anesthetized rats. The microdialysate that perfused from the animal at a flow rate of 0.5 microL/min was on-line transferred to the CMD to remove the high-sodium matrix, followed by ICPMS measurement. The role of the CMD in this on-line system was investigated in detail. With prior addition of EDTA to the microdialysate to form anionic complexes of the metal analytes and the use of NH4Cl as a regenerant to exchange Na(+) with NH4(+) ions, both quantitative recovery of the trace metal analytes and quantitative removal of the sodium matrix could be achieved. Two experimental modes of the monitoring system were constructed. For those metals (e.g., Cu, Zn, and Mn) that existed at (sub)nanogram-per-milliliter concentrations in the microdialysate, the temporal resolution was 10 min when using a 10 microL loop for sample collection, followed by CMD and ICPMS; for those elements (e.g., Ca and Mg) that existed at microgram-per-milliliter levels (or greater), near-real-time analysis was possible because the microdialysate could be led, bypassing the sample loop, directly to the CMD for desalting without any time delay. Further improvement of the temporal resolution for the low-concentration elements was not possible without decreasing the detection limits of mass detection. Among the eight trace metals tested using this on-line system, the method detection limits for Cu, Zn, Mn, Co, Ni, and Pb reached subnanogram-per-milliliter levels; for electrolyte species such as Ca and Mg, the detection limits were in the range of 50-100 ng/mL. Analytical accuracy, expressed as spike recovery, was 100% +/- 15% for all of the elements tested. We demonstrate the applicability of the proposed system through the successful measurement of the basal values of Ca, Mg, Cu, Zn, and Mn in the ECF of a living rat brain and through in vivo monitoring of the concentration profiles of Mn and Pt in the ECF after the injection of drugs (MnCl2 and cisplatin) into the rats. This microdialysis system is the first to offer real-time, in vivo monitoring of trace elements such as Ca and Mg.

Palladium-catalyzed tandem dimerization and cyclization of acetylenic ketones: a convenient method for 3,3'-bifurans using PdCl2(PPh3)2.

Alkynones undergo tandem dimerization and cyclization in the presence of PdCl2(PPh3)2 and triethylamine in tetrahydrofuran at room temperature to give 3,3'-bifurans predominantly. Other palladium catalysts while under similar conditions, by rearrangement, lead to 2,5-disubstituted furans. This distinguished property of PdCl2(PPh3)2 has been attributed to the involvement of hydridopalladium halide. This method provides a simpler route to a variety of furans and a regioselective synthesis of polysubstituted 3,3'-bifurans using easily accessible acetylenic ketones.

Simultaneous supercritical fluid extraction and chemical derivatization for the gas chromatographic-isotope dilution mass spectrometric determination of amphetamine and methamphetamine in urine.

An in-situ supercritical fluid extraction (SFE) and chemical derivatization (ChD) procedure followed by gas chromatography-isotope dilution mass spectrometry (GC-MS) for the determination of amphetamines in urine is described and evaluated. While using celite as the SFE wet-support, the one-pot sample pretreatment procedure also employs ammonium water to alkalize the urine matrix that contains protonated amphetamine (AP) and methamphetamine (MA). The mean recoveries achieved by simultaneous SFE-ChD, i.e., 95% (RSD=3.8%) for AP and 89% (RSD=4.0%) for MA, are significantly better than the corresponding overall recoveries obtained upon stepwise SFE-ChD, suggesting the unreacted trifluoroacetic anhydride (TFA) in the former procedure has strengthened the extracting power of CO, fluid as has been evidenced by a control test. As to GC-MS analysis, the optimal qualitative ions and quantitative ions of the respective analytes were determined via a rigorous evaluation process. Thus, the regression calibration curves for AP and MA in urine are linear within 100 approximately 50,000 ng/ml, with correlation coefficients typically exceeding 0.999. The limits of detection determined by two methods for AP and MA vary from 19 to 50 ng/ml, and limits of quantitation from 21 to 100 ng/ml. Precisions calculated for the triplicate analyses of AP and MA in a 500-ng/ml spiked control, two real-case samples and two quasi real-case samples, respectively, using regression calibration are typically below 10%. The method is simple and reliable. It may serve as an alternative to the existing confirmatory protocol for forensic urine drug testing.

Estimation of metal and organochlorine pesticide exposures and potential health threat by consumption of oysters in Taiwan.

Pollutant concentrations detected in oysters from 12 different culture areas of Taiwan (especially for the Hsiangshan area and the Machu Islands) from 1991-98 were evaluated to investigate potential carcinogenic (inorganic As and organochlorine pesticides) and non-carcinogenic (Cu, Zn, Cd and inorganic As) risk to the public from ingestion of the oysters. The highest geometric mean (GM) Cu and Zn concentrations of 1108 (range 113-2806) and 1567 (range 303-3593) microg/g dry weight were obtained in oysters from the Hsiangshan coastal area. The maximum GM Cd and As concentrations of 6.82 and 19.3 microg/g dry weight were found in oysters from the Machu Islands area. The p,p'-DDE values range from not detectable in Penghu Islands' oysters to 164 ng/g dry weight in Machu Islands' oysters. The highest tDDT (sum of p,p'-DDE, p,p'-DDD and p,p'-DDT) concentrations of 337 and 340 ng/g dry weight were found in oysters from Kimmen and Machu Islands, respectively. A calculated target hazard quotient (THQ; daily intake/reference dose) of 11.4 (based on 139 g oysters/day) for Cu caused by consuming oysters from the Hsiangshan area is higher than that from other areas (range 0.124-5.95). The highest average Cu intake from Hsiangshan's oysters for individuals is 11.4 times (i.e. THQ=11.4) more than that of reference dose (40 microg/kg/day). However, the maximum THQ values for Cd and As caused by consuming oysters collected from the Machu Islands were 5.57 and 2.63 for Cd and As, respectively. Generally, the results of THQ showed that if only the maximally exposed individuals were considered, the value of 65.4% for oyster was higher than 1.0 in comparison with reference dose. All cancer risk estimates for inorganic As from consuming oysters were higher than 10(-6) (range from 128x10(-6) to 509x10(-6) for maximally exposed individuals and range from 17.1x10(-6) to 68.0x10(-6) for typically exposed individuals, respectively); that is the risk of the lower end of the range of acceptable risk. The highest risk estimate for inorganic As was 509x10(-6) for consumption of oysters by Machu Islands' residents. The lifetime cancer risks of 19.0x10(-6) for tDDT by consuming oysters from the Machu Islands was higher than those from the Penghu Islands (0.37x10(-6)). Therefore, the sum of lifetime cancer risks for tDDT and inorganic As had the highest risks (total risk=528x10(-6)) of consuming oysters from the Machu Islands. Furthermore, a 10(-6) upper limit on lifetime risk as the health protection standard would require maximum oyster consumption rates of approximately 0.26 g/day.

Supercritical fluid extraction and clean-up of organochlorine pesticides in Chinese herbal medicine.

A method involving the simultaneous extraction and clean-up of 13 organochlorine pesticides (OCPs) from Chinese herbal medicines (CHMs) was developed using supercritical fluid extraction (SFE) followed by gas chromatography-electron capture detection and mass spectrometric confirmation. The pesticides in the study consisted of alpha-, beta-, gamma-, and delta-benzene hexachloride, heptachlor, aldrin, heptachlor epoxide, endosulfan I, 4,4'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethene), dieldrin, endrin, 4,4'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane), endosulfan II, 4,4'-DDT (2,2-bis(p-chlorophenyl)1,1,1-trichloroethane), endrin aldehyde, and endosulfan sulfate. A series of experiments was conducted to optimize the final extraction conditions [pure CO2, 250 atm extraction pressure (1 atm = 101,325 Pa), 50 degrees C extraction temperature, 5 min static extraction time, 20 min dynamic extraction time, 2.0-g Florisil sorbent on top of 0.1-g samples, 12-ml n-hexane eluting at 1 ml/min, and a 10-ml extraction vessel]. Florisil sorbent was placed with the sample in the SFE vessel to provide a facile and effective clean-up approach. Mean recoveries of 78-121% with reproducibilities of 5-31% were obtained for the pesticides except for endosulfan II, endosulfan sulfate and endrin aldehyde. The simple and rapid method may be used to determine OCPs in CHMs routinely, and in fact, was used to analyze CHMs sold in Taiwan.

Quantitative analysis of antibiotics by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Comparative studies of the matrix-assisted laser desorption/ionization (MALDI) of 30 antibiotics were made using alpha-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin and meso-tetra(N-methyl-4-pyridyl)porphyrin matrices. Most antibiotics generated intense protonated molecules in HCCA and DHB matrices, and sodium or potassium adduct ions in porphyrin matrices. Using sulfonamide antibiotics as model compounds, DHB was found to be the most suitable matrix for quantitative analysis. Detection limits were about 1 picomole. Linear correlation (R2 > 0.97), between the sample quantity over the range 1 to 100 picomole and the signal response, was obtained when ratios of the sum of peak areas of protonated molecules and sodium adduct ions, with reference to that of a structurally analogous internal standard (acetaminophen), were measured. The precision was found to be in the range of 4 to 32% relative standard deviation, dependent on the type and concentration of the analyte. A simple acylation derivatization process was developed to confirm the presence of suspected antibiotic residues. It is demonstrated that MALDI is a viable technique for the quantitative analysis of low molecular weight antibiotics.

Reassessment of PCDD/DFs and Co-PCBs toxicity in contaminated rice-bran oil responsible for the disease "Yu-Cheng".

Reassessment of dibenzo-p-dioxins and dibenzofurans (PCDD/DFs) and coplanar polychlorinated biphenyls (Co-PCBs) in toxic Yu-Cheng rice-bran oil was carried out. The contaminating source appears to be a mixture of KC-400 and KC-500. The oil contains 176 micrograms/g PCBs and 37.194 ng/g 2,3,7,8,-TCDD toxicity equivalency (TEQ). The TEQ contributions from the PCDDs (4.472 ng/g), PCDFs (19.568 ng/g), and Co-PCBs (13.154 ng/g) are 12% 53% and 35%, respectively. Complete identification and quantitation suggests that 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 2,3,7,8,-TCDF. 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and PCB# 77 are candidates for the primary causal agents of Yu-Cheng disease.

Supercritical fluid extraction and clean-up of organochlorine pesticides and polychlorinated biphenyls in mussels.

The simultaneous extraction and clean-up of mussel samples followed by gas chromatography with electron-capture detection and mass spectrometric confirmation of 15 organochlorine pesticides (OCPs) and 11 polychlorinated biphenyls (PCBs) is developed using Florisil sorbent in the supercritical fluid extraction cell. The method detection limits vary from 1 to 10 ng/g for OCPs and from 2 to 15 ng/g for PCBs. Mean reproducibilities of 11% and 10% and mean recoveries of 80% and 53%, respectively, for OCPs and PCBs are obtained. The feasibility of the proposed supercritical fluid extraction method was confirmed by analyzing a certified reference material and mussels collected from Taiwan region. The method is simple, rapid and requires only small amounts of samples and solvents. It may serve as a screening protocol for the determination of OCPs and PCBs in mussels on a routine basis.

Application of hemodilution in microsurgical free flap transplantation.

The intra-operative hemodilution and blood auto-transfusion is a blood-saving technique that can be performed when major blood loss is expected. The effects of this technique were studied in 30 microsurgical free flap transplantation patients. Between 400-600 ml blood was collected from the patients before surgery. The patients received dextran, a balanced salt solution, and glucose with the ratio of 3:1 to the collected blood volume, bleeding, and urine before the blood auto-transfusion. There were no significant changes in RBC, HCT, MCV, blood pressure, or heart rate. Of the 30 free flaps, 28 were successful with a 93.3% survival rate. The safety of intra-operative hemodilution and autologous blood transfusion in microsurgery as well as the effect of hemodilution on transplanted flap survival are discussed in this study.

PCDD/DFs and coplanar PCBs in sediment and fish samples from the Er-Jen river in Taiwan.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/DFs) and coplanar polychlorinated biphenyls (Co-PCBs) have been determined in sediment and fish samples from the Er-Jen river in Taiwan. Sediment samples collected near the incineration site show TEQ of PCDD/DFs ranging from 14 pg/g to 14200 pg/g and 2,3,7,8-tetrachlorodibenzo-p-dioxins equivalents (TCDD-EQ) of Co-PCBs ranging from 9 pg/g to 1030 pg/g. The fish samples show TEQ of PCDD/DFs ranging from 37 pg/g to 2084 pg/g and TCDD-EQ of Co-PCBs ranging from 12 pg/g to 120 pg/g. Possible PCDD/DFs sources are discharged PCBs, open-air incineration activities, and pentachlorophenol. PCB#126 was found to degrade more readily than PCB#77. TCDD-EQ of Co-PCBs could be estimated from the total concentration of PCBs. A daily intake value of 28 pg/kg/day to 450 pg/kg/day was estimated when consuming fish caught from the Er-Jen river.

Multi-residue matrix solid-phase dispersion method for the determination of six synthetic pyrethroids in vegetables followed by gas chromatography with electron capture detection.

An effective multi-residue matrix solid-phase dispersion (MSPD) extraction and gas chromatographic-electron-capture detection method for the determination of six synthetic pyrethroids (fenpropathrin, cyhalothrin, permethrin, cypermethrin, fenvalerate and deltamethrin) in 5 g of vegetables (West Indian gherkin, eggplant, pak-choi, cabbage and garden peas) is described. The method uses a Florisil-based MSPD column for direct in-line clean-up with n-hexane-acetone (9:1). Recoveries calculated at 0.1 and 0.5 micrograms/g fortification levels were between 92 and 113%. The method detection limits were between 5.1 and 91.5 ng/g. The method compared favourably with the traditional method in terms of the sample size, analysis time and overall cost. The method may serve as a screening protocol for the determination of pyrethroids in vegetables.

Matrix solid-phase dispersion extraction and gas chromatographic screening of polychlorinated biphenyls in fish.

A matrix solid-phase extraction (MSPD) and gas chromatographic-electron-capture detection method for determining polychlorinated biphenyls (PCBs) in fish (grass carp) is described. The method uses an octadecylsilyl-derivatized silica and acidic silica gel-based MSPD co-column for direct in-line clean-up. The performance of the method compares favourably with that of the traditional approach but the sample size, analysis time and overall cost are lower. The method may serve as a screening protocol for the isolation and determination of PCBs in fish.

Clinical experience and surgical anatomy of 32 free anterolateral thigh flap transplantations.

The anterolateral thigh flap, first described by Song, is one of the septocutaneous flaps in the thigh. Since November 1985 we have transferred 32 anterolateral thigh flaps successfully without any loss. Six of these flaps were performed in Australia. The vascular pedicles are divided into three types: fasciocutaneous perforator (37.5%-12/32), vertical musculocutaneous perforator (56.2%-18/32) and horizontal musculocutaneous perforator (6.6%-2/32). This paper presents the surgical anatomy of the flap and clinical experience.

Quantitative imaging of boron, calcium, magnesium, potassium, and sodium distributions in cultured cells with ion microscopy.

A method for the conversion of intensity information in ion micrographs of freeze-fractured, freeze-dried cultured cells to local dry weight elemental concentrations is presented. Homogenates generated from cultured cells are used as calibration standards. Ion microscope (IM) relative sensitivity factors for B, Ca, K, Mg, and Na with respect to the matrix element C are determined by the correlation of IM and inductively coupled plasma atomic emission spectrometry analyses of the cellular homogenates. After calibration of the IM imaging system, the relative sensitivity factors are used to determine local intracellular concentrations of B, Ca, K, Mg, and Na in cultured Swiss 3T3 fibroblasts. Intracellular B was introduced through cellular uptake of Na2B12H11SH, a candidate therapeutic agent for boron neutron capture cancer therapy. The IM intracellular concentration results show good agreement with published electron probe X-ray microanalysis results. Estimated detection limits are in the low- to subparts-per-million dry weight concentration range.

Experience using microvascular anastomotic rings for the transplantation of 20 free flaps.

A new microvascular anastomosis technique was employed with success in 1985-1986 with the result of 18 free-flap survivals and 2 failures. The rate of success in microvascular anastomoses was 90.5 percent (38 of 42). The advantages of this technique are its simplicity, convenience, and safety, and it may shorten the duration of the operation. Our experience in using vascular anastomotic rings is presented.

Quantitative imaging of free and total intracellular calcium in cultured cells.

Techniques of fluorescence and ion microscopies were combined to study the free [Ca2+] and total Ca in NIH 3T3 fibroblast and L6 rat myoblast cells. Free Ca2+ measurements with the Ca2+ indicator fura-2 and digital imaging reveal an inhomogeneous distribution of free cytoplasmic Ca2+ in both cell lines. Fura-2 also reveals a difference in free Ca2+ activity between the nucleus and cytoplasm of cells. Ion microscopic observations on sister cells show that total Ca in the cytoplasm is also inhomogeneously distributed and that mean cytoplasmic levels of total Ca are higher than levels in the nuclei. In the nuclei of NIH 3T3 cells, the mean free [Ca2+] and total [Ca] were 110 +/- 30 nM and 225 +/- 43 microM, respectively, while regions in the cell cytoplasm contained up to 490 +/- 270 nM free [Ca2+] and 559 +/- 184 microM mean total [Ca]. Intracellular total Ca was greater than 3 orders of magnitude higher than intracellular free Ca2+ in either nuclear or cytoplasmic compartments. Perinuclear cytoplasmic regions in 3T3 cells contained higher free and total Ca than the cell nucleus. Loading of cells with fura-2 did not modify the subcellular distribution of total K, Na, Ca, or Mg. This combination of two powerful ion imaging techniques provides a comparison between free and total calcium in cells and introduces a different approach for examining the role of this important element in cell physiology.