Biological reference materials for assaying human albumin in urine

Summary To assess the interlaboratory variation in the results of albumin measurements, we prepared albumin solutions in human urine at various concentrations within the normal range. Since some investigators have reported that albumin is unstable in some human urine samples stored at –20°C, we screened urine samples from 21 persons to identify samples that were stable under these conditions and that had low native albumin content. The urine of two donors met these criteria, and they provided urine, which we prefiltered, sterile-filtered, and spiked with commercially available human serum albumin. The albumin was characterized as pure by a Lowry assay of protein content with National Institute of Standards and Technology bovine serum albumin (standard reference material 926) as the standard and by the appearance of one band on agarose gel electrophoresis. To evaluate the necessity for additional stabilization when urine samples are stored at –20°C, a surfactant was included in one set of materials and not included in another. The materials with surfactant have been evaluated for 10.5 months and those without surfactant for 5 months. The preserved materials showed no significant loss of activity during this period. The unpreserved materials remained stable for 2 months, and then the two higher level materials appeared to loose activity. The negative slope of the highest level of unpreserved material was statistically significant (p=0.01) during this period. In our laboratory, the albumin recovered by enzyme immunoassay was 106.7% and 115.9% in two preserved normal-range materials and 102.2% and 106.3% in similar unpreserved materials.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Synthesis,Structure and Reactivity of Organoindium 1,2-Benzenedithiolates and 2-Amidobenzenethiolates

Organoindium compounds of redox active 1,2-benzenedithiolate and 2-amidobenzenethiolate ligands were synthesized and tested for reactivity against mild oxidants. The reaction of Me₃In and (NCN)InMe₂ [NCN=2,6-bis(dimethylaminomethy)phenyl] with 3,4-toluenedithiol (H₂tdt) at room temperature afforded [MeIn(tdt)(py)]₂ ( 1 ) and (NCN)In(tdt) ( 2 ), respectively. A similar reaction of Me₃In with 2-aminobenzenethiol (H₂abt) in toluene under reflux afforded [MeIn(abt)(py)]₂ ( 3 ). The reaction of (NCN)InCl₂ with one equivalent of Li₂(abt) or two equivalents of Li(Habt) afforded the compounds [(NCN)In(abt)]⋅LiCl(thf)₂ ( 4 ⋅LiCl(thf)₂) and (NCN)In(Habt)₂ ( 5 ), respectively. The X-ray crystal structures of 1 and 3 are similar and show dimeric structures via μ-S-(tdt) and μ-N-(abt) ligands, respectively. Compounds 2 and 4 possess similar monomeric structures and tridentate NCN pincer ligands. DFT computational studies have been used to rationalize the observed solid-state structures and discern the potential reactivity of compounds 1 – 4 against oxidants. The reaction of 1 and 2 with excess iodine resulted in loss of the 3,4-toluenedithiolate ligand and the formation of the oligomeric disulfide [tdt]_n, while 3 and 4 showed no reactivity under similar conditions. This contrasts the reactivity of previously reported organoindium o-amidophenolate complexes which undergo oxidative addition of iodine to afford ligand-centered radical species.     

Determination of beta-agonist residues in bovine urine using liquid chromatography-tandem mass spectrometry

A multiresidue method was developed and validated to screen bovine urine samples for 10 beta-2-adrenergic agonistic drugs--brombuterol, cimaterol, clenbuterol, clenpenterol, isoxsuprine, mabuterol, ractopamine, ritodrine, salbutamol, and tulobuterol--at the 2 microg/L level. The method is also quantitative in the range of 1 to 4 microg/L for all analytes except salbutamol. The procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on solid-phase extraction columns, followed by detection using a liquid chromatograph-tandem quadrupole mass spectrometer operated in the positive-ion atmospheric pressure chemical ionization multiple-reaction monitoring mode. Method validation included assessment of recoveries, repeatabilities, linearity of responses, decision limits, and detection capabilities. Overall average recoveries ranged from 70-91%; recoveries were generally lower for salbutamol. The decision limits ranged from 0.4-1.0 microg/L, and detection capabilities from 0.6-1.7 microg/L.     

Three 18-Electron Tantalum(I) Compounds, TaCl(CO)(2)(dppe)(2), [TaCl(CO)(2)(dppe)(2)](2x), and TaCl(CO)(4)(dppe) (dppe = 1,2-Bis(diphenylphosphino)ethane), Which Exhibit Low Levels of Paramagnetism

Reductive carbonylation of TaCl(5) in the presence of 1,2-bis(diphenylphosphino)ethane (dppe) under the appropriate conditions results in the formation of TaCl(CO)(2)(dppe)(2) (1), as the major product, and the possibly cyclic oligomer [TaCl(CO)(2)(dppe)(2)](2)(x)() (2, 2x >/= 4) as a minor product. Carbonylation of 1 (1 atm) results in the rapid but reversible formation of TaCl(CO)(4)(dppe) (3). Solutions of all three compounds exhibit low levels of paramagnetism, possibly attributable to thermal population of low-lying triplet excited states. Crystal data for the toluene solvate of 1, C(68)H(64)ClO(2)P(4)Ta: triclinic, P&onemacr; (No. 2), a = 13.937(12) ?, b = 14.811(7) ?, c = 14.929(9) ?, alpha = 102.30(5) degrees, beta = 95.60(7) degrees, gamma = 98.41(5) degrees, Z = 2.     

Alternative methodology for the analysis of progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle

Research has shown that traditional solvent extraction procedures used for the analysis of endogenous steroids often give inconsistent recoveries and results. However, a single-laboratory validation of a liquid chromatography/tandem mass specrometry method using 2 product ions per transition for progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle showed accuracy and precision to within 23% at concentrations ranging from 0.5 to 2.0 microg/kg. Homogenized samples were pretreated with methanol to denature endogenous enzymes. Following removal of methanol, samples were treated overnight with Helix pomatia beta-glucuronidase to deconjugate glucuronide conjugates. Alkali digestion of the samples in KOH solutions was done under shaking at 37 degrees C for 30 min. The digestate was extracted with methyl tert-butyl ether, and the extracts were cleaned by partitioning between acetonitrile-hexane, followed by solid-phase extraction cleanup on silica cartridges. In bovine liver, average recoveries exceeded 54% for all analytes, and the within-run assay coefficients of variations were < 6 and 13% for high (2.0 microg/kg) and low (0.3 microg/kg) analyte concentrations, respectively. In veal muscle, average recoveries exceeded 60%, and the analysis of blind spikes gave accuracy estimates of over 85%, with coefficients of variation (CVs) < 15% for all analytes. The CVs for the multiple reaction monitoring ion ratios for all compounds were < 22% for all validation data. The method meets the requirements for confirmatory methods as outlined in 2002/657/EC. An analyst is capable of processing up to 20 samples within 5 days.     

Characterisation of structural changes in collagen with Raman spectroscopy

Raman spectroscopy can detect conformational changes in collagen structures and these findings are reviewed in this article. More specifically, some progressive diseases are mainly caused by alterations of collagen molecules but what is occurring at the biochemical level of this complex molecule usually remains unclear. While it may be true that a number of analytical techniques can analyze collagen, most of them have a series of limitations that limit their applicability to a wide range of samples. To understand in more detail the progression of a disease due to changes in the collagen structure, a technique that can detect subtle alterations at the biochemical level is needed. Raman spectroscopy is a label-free and noninvasive technique that can easily pick up on any conformational changes reflected primarily at the lipids, amides and proline and hydroxyproline regions. This review is the first compilation of studies of conformational changes in collagen molecules, providing help to understand changes in collagen biochemistry that can be of relevance to the human wound healing, ageing and pathologies.     

Imaging cytoplasmic cAMP in mouse brainstem neurons

Background  

cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca²⁺ levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before.     

Stable isotopic studies of earthworm feeding ecology in tropical ecosystems of Puerto Rico

Feeding strategies of earthworms and their influence on soil processes are often inferred from morphological, behavioral and physiological traits. We used (13)C and (15)N natural abundance in earthworms, soils and plants to explore patterns of resource utilization by different species of earthworms in three tropical ecosystems in Puerto Rico. In a high altitude dwarf forest, native earthworms Trigaster longissimus and Estherella sp. showed less (15)N enrichment ((15)N = 3-6 per thousand) than exotic Pontoscolex corethrurus ((15)N =7-9 per thousand) indicating different food sources or stronger isotopic discrimination by the latter. Conversely, in a lower altitude tabonuco forest, Estherella sp. and P. corethrurus overlapped completely in (15)N enrichment ((15)N = 6-9 per thousand), suggesting the potential for interspecific competition for N resources. A tabonuco forest converted to pasture contained only P. corethrurus which were less enriched in (15)N than those in the forest sites, but more highly enriched in (13)C suggesting assimilation of C from the predominant C(4) grass. These results support the utility of stable isotopes to delineate resource partitioning and potential competitive interactions among earthworm species. Copyright 1999 John Wiley & Sons, Ltd.