Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns.
(1/113)
OBJECTIVES: To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl(-)) on plasma unbound bilirubin (B(f)) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. DESIGN AND METHODS: B(f) was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with B(f) measured at a 2-fold sample dilution using a FloPro Analyzer. B(f) was measured at two peroxidase concentrations to determine whether the peroxidase steady state B(f) (B(fss)) measurements were significantly less than the equilibrium B(f) (B(feq)), in which case it was necessary to calculate B(feq) from the two B(fss) measurements. B(f) was also measured before and after adding 100 mmol/L Cl(-) to the UB Analyzer assay buffer. RESULTS: B(feq) at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with B(feq) at the 2-fold dilution (mean 8.2+/-5.2 nmol/L versus 73.5+/-70 nmol/L, respectively, p<0.0001; correlation r=0.6). The two UB Analyzer B(fss) measurements were significantly less than B(feq) in 42 of 74 (57%) samples, and Cl(-) increased B(feq) in 66 of 74 (89%) samples by a mean of 82+/-67%. CONCLUSIONS: B(fss) measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl(-) and a single peroxidase concentration is significantly less than the B(feq) in undiluted plasma. Accurate B(f) measurements can be made only in minimally diluted serum or plasma. (+info)
Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.
(2/113)
Using cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA+ RNA. Aiming to use small quantities of solid tissue samples (10-20 mg/sample), polyA+ RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen-frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses. (+info)
Metabolism, pharmacokinetics, and excretion of a nonpeptidic substance P receptor antagonist, ezlopitant, in normal healthy male volunteers: characterization of polar metabolites by chemical derivatization with dansyl chloride.
(3/113)
The excretion, biotransformation, and pharmacokinetics of ezlopitant [(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)-amine ], a substance P receptor antagonist, were investigated in healthy male volunteers after oral administration of a single 200-mg (approximately 93 microCi/subject) dose of [(14)C]ezlopitant. The total recovery of administered radioactive dose was 82.8 +/- 5.1, with 32.0 +/- 4.2% in the urine and 50.8 +/- 1.4% in the feces. Mean observed maximal serum concentrations for ezlopitant and total radioactivity were achieved at approximately 2 h after oral administration; thus, ezlopitant was rapidly absorbed. Ezlopitant was extensively metabolized in humans, since no unchanged drug was detected in urine and feces. The major pathway of ezlopitant in humans was the result of the oxidation of the isopropyl side chain to form the omega-hydroxy and omega-1-hydroxy (M16) metabolites. M16 and omega,omega-1-dihydroxy (1,2-dihydroxy, M12) were identified as the major circulating metabolites accounting for 64.6 and 15.4% of total circulating radioactivity, respectively. In feces, the major metabolite M14 was characterized as the propionic acid metabolite and formed by further oxidation of the omega-hydroxy metabolite. The urinary metabolites were the result of cleaved metabolites caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. The metabolites (M1A, M1B, and M4), approximately 34% of the total radioactivity in urine, were identified as benzyl amine derivatives. These were polar metabolites that were further characterized using the reaction with dansyl chloride to derivatize the primary amines and phenol moieties to less polar analytes. The other metabolites were the result of O-demethylation, dehydrogenation of the isopropyl group, and oxidation on the quinuclidine moiety. (+info)
Arsenic determination in marine sediment using ultrasound for sample preparation.
(4/113)
This work deals with As determination in marine sediment using ultrasound for sample preparation. It is shown that As can be quantitatively extracted from marine sediment using 20% (v/v) HCl and sonication. The slurry is centrifuged and the analyte is determined in the supernatant by hydride generation atomic absorption spectrometry (HG AAS). A flow injection (FI) system is employed for hydride generation, with 0.5% (m/v) NaBH(4) used as reducdant and a 20% (v/v) HCl used as sample carrier. The limit of quantification is 1.6 microg g(-1) of As, which is based on 800 microl of sample solution and 0.200 g of sample mass in a volume of 50 mL. Certified and non certified marine sediment samples were analyzed; the results were in accordance with the certified or reference values. Speciation analysis by HPLC-ICP-MS showed that As(V) is the only detectable As species present in the supernatant of the centrifuged sample. (+info)
On-plate digestion using a commercial microfraction collector for nano-HPLC matrix-assisted laser desorption/ionization tandem time-of-flight protein analysis.
(5/113)
Synthesis of bis(amino alcohol)oxalamides and their usage for the preconcentration of trace metals by cloud point extraction.
(6/113)
C(2)-Symmetric two bis(amino alcohol)oxalamides (diamidediols) were synthesized and fully characterized. A new method was developed and successfully applied for the simultaneous preconcentration of both trace and toxic metals in water, by using C(2)-symmetric compounds. Under the optimum experimental conditions (i.e. pH = 10.0 +/- 0.2, 2.75 x 10(-3) mol L(-1) N,N'-bis[(1R)-1-ethyl-2-hydroxyethyl]ethanediamide (DAD1), 1.75 x 10(-3) mol L(-1) N,N'-bis[(1S)-1-benzyl-2-hydroxyethyl]-ethanediamide (DAD2), 0.10% w/v octylphenoxy-polyethoxyethanol (Triton X-114)), calibration graphs were linear in the range of 2.5 - 25.0 ng mL(-1) for Cu and Cd, 5.0 - 25.0 ng mL(-1) for Co and Ni. The enrichment factors were 18, 23, 18 and 20 for Cd, Cu, Co and Ni in the case of DAD1, respectively; 20, 22, 17 and 20 for Cd, Cu, Co and Ni in the case of DAD2. The limits of detection for DAD1 were found to be 0.45, 0.50, 1.25 and 0.60 ng mL(-1) for Cd, Cu, Co and Ni, respectively, and for DAD2 were found to be 0.44, 0.25, 0.60 and 1.55 ng mL(-1) for Cd, Cu, Co and Ni, respectively. The developed method was applied to the determination of Cu, Cd, Co and Ni in water samples and certified reference materials with satisfactory results. (+info)
Triple-phase single-drop microextraction of silver and its determination using graphite-furnace atomic-absorption spectrometry.
(7/113)
A new method is described for the determination of silver based on triple-phase microextraction using diethyldithio-carbamate (DDTC) and thioaminophenol. Ag is separated and preconcentrated from the matrix of the sample solution, and finally determined by electrothermal atomic-absorption spectroscopy. The parameters that affect the efficiency were investigated. Under the optimized conditions, a 30-fold preconcentration factor with a detection limit of 0.05 microg L(-1) was achieved. The relative standard deviation was 10% (5 determinations). The developed method was applied to the determination of trace Ag in water samples. (+info)
Moving known libraries to an addressable array: a site-selective hetero-Michael reaction.
(8/113)