A new method for the determination of plutonium and americium using high pressure microwave digestion and alpha-spectrometry or ICP-SMS

Plutonium and americium are radionuclides particularly difficult to measure in environmental samples because they are alpha-emitters and therefore necessitate a careful separation before any measurement, either using radiometric methods or ICP-SMS. Recent developments in extraction chromatography resins such as Eichrom (R) TRU and TEVA have resolved many of the analytical problems but drawbacks such as low recovery and spectral interferences still occasionally occur. Here, we report on the use of the new Eichrom (R) DGA resin in association with TEVA resin and high pressure microwave acid leaching for the sequential determination of plutonium and americium in environmental samples. The method results in average recoveries of 83 +/- 15% for plutonium and 73 +/- 22% for americium (n = 60), and a less than 10% deviation from reference values of four IAEA reference materials and three samples from intercomparisons exercises. The method is also suitable for measuring Pu-239 in water samples at the mu Bq/l level, if ICP-SMS is used for the measurement.

Clinical evaluation of a method for detecting superficial surgical transitional cell carcinoma of the bladder by light-induced fluorescence of protoporphyrin IX following the topical application of 5-aminolevulinic acid: preliminary results.

BACKGROUND AND OBJECTIVE: In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of "flat" urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5-aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: Several hours after bladder instillation of an aqueous solution of ALA in 34 patients, a Krypton ion laser or a filtered Xenon arc-lamp was used to excite PpIX fluorescence. Tissue samples for histological analysis were taken while observing the bladder wall either by means of a video camera, or by direct endoscopic observation. RESULTS: A good correlation was found between the PpIX fluorescence and the histopathological diagnosis. On a total of 215 biopsies, 143 in fluorescent and 72 in nonfluorescent areas, all visible tumors on white light cytoscopy appeared in a bright red fluorescence with the photodetection technique. In addition, this method permitted to discover 47 unsuspected carcinomatous lesions on white light observation, among which 40% were carcinoma in situ. CONCLUSION: PpIX fluorescence induced by instillation into the bladder of 5-ALA is an efficient method of mapping the mucosa in bladder carcinoma.

Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.

A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency.

Once-yearly zoledronic acid for treatment of postmenopausal osteoporosis.

BACKGROUND: A single infusion of intravenous zoledronic acid decreases bone turnover and improves bone density at 12 months in postmenopausal women with osteoporosis. We assessed the effects of annual infusions of zoledronic acid on fracture risk during a 3-year period. METHODS: In this double-blind, placebo-controlled trial, 3889 patients (mean age, 73 years) were randomly assigned to receive a single 15-minute infusion of zoledronic acid (5 mg) and 3876 were assigned to receive placebo at baseline, at 12 months, and at 24 months; the patients were followed until 36 months. Primary end points were new vertebral fracture (in patients not taking concomitant osteoporosis medications) and hip fracture (in all patients). Secondary end points included bone mineral density, bone turnover markers, and safety outcomes. RESULTS: Treatment with zoledronic acid reduced the risk of morphometric vertebral fracture by 70% during a 3-year period, as compared with placebo (3.3% in the zoledronic-acid group vs. 10.9% in the placebo group; relative risk, 0.30; 95% confidence interval [CI], 0.24 to 0.38) and reduced the risk of hip fracture by 41% (1.4% in the zoledronic-acid group vs. 2.5% in the placebo group; hazard ratio, 0.59; 95% CI, 0.42 to 0.83). Nonvertebral fractures, clinical fractures, and clinical vertebral fractures were reduced by 25%, 33%, and 77%, respectively (P<0.001 for all comparisons). Zoledronic acid was also associated with a significant improvement in bone mineral density and bone metabolism markers. Adverse events, including change in renal function, were similar in the two study groups. However, serious atrial fibrillation occurred more frequently in the zoledronic acid group (in 50 vs. 20 patients, P<0.001). CONCLUSIONS: A once-yearly infusion of zoledronic acid during a 3-year period significantly reduced the risk of vertebral, hip, and other fractures. (ClinicalTrials.gov number, NCT00049829.)

An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Microbiological diagnosis of community-acquired respiratory tract infections by nucleic acid detection.

Background: Microbiological diagnostic procedures have changed significantly over the last decade. Initially the implementation of the polymerase chain reaction (PCR) resulted in improved detection tests for microbes that were difficult or even impossible to detect by conventional methods such as culture and serology, especially in community-acquired respiratory tract infections (CA-RTI). A further improvement was the development of real-time PCR, which allows end point detection and quantification, and many diagnostic laboratories have now implemented this powerful method. Objective: At present, new performant and convenient molecular tests have emerged targeting in parallel many viruses and bacteria responsible for lower and/or upper respiratory tract infections. The range of test formats and microbial agents detected is evolving very quickly and the added value of these new tests needs to be studied in terms of better use of antibiotics, better patient management, duration of hospitalization and overall costs. Conclusions: Molecular tools for a better microbial documentation of CA-RTI are now available. Controlled studies are now required to address the relevance issue of these new methods, such as, for example, the role of some newly detected respiratory viruses or of the microbial DNA load in a particular patient at a particular time. The future challenge for molecular diagnosis will be to become easy to handle, highly efficient and cost-effective, delivering rapid results with a direct impact on clinical management.

A dendritic cell vaccine pulsed with autologous hypochlorous Acid-oxidized ovarian cancer lysate primes effective broad antitumor immunity: from bench to bedside.

PURPOSE: Whole tumor lysates are promising antigen sources for dendritic cell (DC) therapy as they contain many relevant immunogenic epitopes to help prevent tumor escape. Two common methods of tumor lysate preparations are freeze-thaw processing and UVB irradiation to induce necrosis and apoptosis, respectively. Hypochlorous acid (HOCl) oxidation is a new method for inducing primary necrosis and enhancing the immunogenicity of tumor cells. EXPERIMENTAL DESIGN: We compared the ability of DCs to engulf three different tumor lysate preparations, produce T-helper 1 (TH1)-priming cytokines and chemokines, stimulate mixed leukocyte reactions (MLR), and finally elicit T-cell responses capable of controlling tumor growth in vivo. RESULTS: We showed that DCs engulfed HOCl-oxidized lysate most efficiently stimulated robust MLRs, and elicited strong tumor-specific IFN-γ secretions in autologous T cells. These DCs produced the highest levels of TH1-priming cytokines and chemokines, including interleukin (IL)-12. Mice vaccinated with HOCl-oxidized ID8-ova lysate-pulsed DCs developed T-cell responses that effectively controlled tumor growth. Safety, immunogenicity of autologous DCs pulsed with HOCl-oxidized autologous tumor lysate (OCDC vaccine), clinical efficacy, and progression-free survival (PFS) were evaluated in a pilot study of five subjects with recurrent ovarian cancer. OCDC vaccination produced few grade 1 toxicities and elicited potent T-cell responses against known ovarian tumor antigens. Circulating regulatory T cells and serum IL-10 were also reduced. Two subjects experienced durable PFS of 24 months or more after OCDC. CONCLUSIONS: This is the first study showing the potential efficacy of a DC vaccine pulsed with HOCl-oxidized tumor lysate, a novel approach in preparing DC vaccine that is potentially applicable to many cancers. Clin Cancer Res; 19(17); 4801-15. ©2013 AACR.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

F+0 renography in neonates and infants younger than 6 months: an accurate method to diagnose severe obstructive uropathy.

We studied the response to F+0 renography and the relative and absolute individual kidney function in neonates and &lt; 6-mo-old infants before and after surgery for unilateral ureteropelvic junction obstruction (UJO). METHODS: The results obtained at diagnosis and after pyeloplasty for 9 children (8 boys, 1 girl; age range, 0.8-5.9 mo; mean age +/- SD, 2.4 +/- 1.5 mo) with proven unilateral UJO (i.e., affected kidney [AK]) and an unremarkable contralateral kidney (i.e., normal kidney [NK]) were evaluated and compared with a control group of 10 children (6 boys, 4 girls; age range, 0.8-2.8 mo; mean age, 1.5 +/- 0.7 mo) selected because of symmetric renal function, absence of vesicoureteral reflux or infection, and an initially dilated but not obstructed renal pelvis as proven by follow-up. Renography was performed for 20 min after injection of (123)I-hippuran (OIH) (0.5-1.0 MBq/kg) immediately followed by furosemide (1 mg/kg). The relative and absolute renal functions and the response to furosemide were measured on background-subtracted and depth-corrected renograms. The response to furosemide was quantified by an elimination index (EI), defined as the ratio of the 3- to 20-min activities: An EI &gt; or = 3 was considered definitively normal and an EI &lt; or = 1 definitively abnormal. If EI was equivocal (1 &lt; EI &lt; 3), the response to gravity-assisted drainage was used to differentiate AKs from NKs. Absolute separate renal function was measured by an accumulation index (AI), defined as the percentage of (123)I-OIH (%ID) extracted by the kidney 30-90 s after maximal cardiac activity. RESULTS: All AKs had definitively abnormal EIs at diagnosis (mean, 0.56 +/- 0.12) and were significantly lower than the EIs of the NKs (mean, 3.24 +/- 1.88) and of the 20 control kidneys (mean, 3.81 +/- 1.97; P &lt; 0.001). The EIs of the AKs significantly improved (mean, 2.81 +/- 0.64; P &lt; 0.05) after pyeloplasty. At diagnosis, the AIs of the AKs were significantly lower (mean, 6.31 +/- 2.33 %ID) than the AIs of the NKs (mean, 9.43 +/- 1.12 %ID) and of the control kidneys (mean, 9.05 +/- 1.17 %ID; P &lt; 0.05). The AIs of the AKs increased at follow-up (mean, 7.81 +/- 2.23 %ID) but remained lower than those of the NKs (mean, 10.75 +/- 1.35 %ID; P &lt; 0.05). CONCLUSION: In neonates and infants younger than 6 mo, (123)I-OIH renography with early furosemide injection (F+0) allowed us to reliably diagnose AKs and to determine if parenchymal function was normal or impaired and if it improved after surgery.

Accuracy profile validation of a new method for carbon monoxide measurement in the human blood using headspace-gas chromatography-mass spectrometry (HS-GC-MS).

The aim of our study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas ((13)CO) formed by the reaction of labeled formic acid formic acid (H(13)COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of (13)CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10 μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.

Fracture risk and zoledronic acid therapy in men with osteoporosis.

BACKGROUND: Fractures in men are a major health issue, and data on the antifracture efficacy of therapies for osteoporosis in men are limited. We studied the effect of zoledronic acid on fracture risk among men with osteoporosis. METHODS: In this multicenter, double-blind, placebo-controlled trial, we randomly assigned 1199 men with primary or hypogonadism-associated osteoporosis who were 50 to 85 years of age to receive an intravenous infusion of zoledronic acid (5 mg) or placebo at baseline and at 12 months. Participants received daily calcium and vitamin D supplementation. The primary end point was the proportion of participants with one or more new morphometric vertebral fractures over a period of 24 months. RESULTS: The rate of any new morphometric vertebral fracture was 1.6% in the zoledronic acid group and 4.9% in the placebo group over the 24-month period, representing a 67% risk reduction with zoledronic acid (relative risk, 0.33; 95% confidence interval, 0.16 to 0.70; P=0.002). As compared with men who received placebo, men who received zoledronic acid had fewer moderate-to-severe vertebral fractures (P=0.03) and less height loss (P=0.002). Fewer participants who received zoledronic acid had clinical vertebral or nonvertebral fractures, although this difference did not reach significance because of the small number of fractures. Bone mineral density was higher and bone-turnover markers were lower in the men who received zoledronic acid (P<0.05 for both comparisons). Results were similar in men with low serum levels of total testosterone. The zoledronic acid and placebo groups did not differ significantly with respect to the incidence of death (2.6% and 2.9%, respectively) or serious adverse events (25.3% and 25.2%). CONCLUSIONS: Zoledronic acid treatment was associated with a significantly reduced risk of vertebral fracture among men with osteoporosis. (Funded by Novartis Pharma; ClinicalTrials.gov number, NCT00439647.).

Comparative effects of losartan and irbesartan on serum uric acid in hypertensive patients with hyperuricaemia and gout.

OBJECTIVE: Losartan has been shown to increase urinary uric acid excretion and hence to lower serum uric acid levels. The purposes of the present study were: (1) to evaluate the effects of losartan on serum uric acid in hypertensive patients with hyperuricemia and gout, (2) to compare the effects of losartan with those of irbesartan, another angiotensin II receptor antagonist and (3) to evaluate whether losartan 50 mg b.i.d. has a greater impact on serum uric acid levels than losartan 50 mg once a day. METHODS: Thirteen hypertensive patients with hyperuricaemia and gout completed this prospective, randomized, double-blind, cross-over study. Uric acid-lowering drugs were stopped 3 weeks before the beginning of the study. Patients were randomized to receive either losartan 50 mg or irbesartan 150 mg once a day, for 4 weeks. During this phase, a placebo was given in the evening. After 4 weeks, the dose was increased to losartan 50 mg b.i.d., or irbesartan 150 mg b.i.d. for another 4 week period. Subsequently, the patients were switched to the alternative treatment modality. Enalapril (20 mg o.d.) was given during the run-in period and between the two treatment phases. Serum and urinary uric acid were measured at the beginning and at the end of each treatment phase. RESULTS: Our results show that losartan 50 mg once daily decreased serum uric acid levels from 538 +/- 26 to 491 +/- 20 micromol/l (P < 0.01). Irbesartan had no effect on serum uric acid. Increasing the dose of losartan from 50 mg o.d. to 50 mg twice a day, did not further decrease serum uric acid. This may in part be due to a low compliance to the evening dose as measured with an electronic device. Indeed, whatever the prescribed drug, the mean compliance of the evening dose was always significantly lower than that of the morning dose. The uricosuric effect of losartan appears to decrease with time when a new steady state of lower serum uric acid is reached. CONCLUSIONS: In contrast to irbesartan, losartan was uricosuric and decreased serum uric acid levels. Losartan 50 mg b.i.d. did not produce a greater fall in serum uric acid than losartan once a day. Losartan might be a useful therapeutic tool to control blood pressure and reduce serum uric acid levels in hypertensive patients with hyperuricaemia and gout.