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PubMed1987

Selected ion monitoring analysis of monoamine metabolites in cerebrospinal fluid. Application to the study of in vivo effects of alpha 2-antagonists.

The technique of isotope dilution mass spectrometry has been used for the measurement of biogenic amine metabolites in cerebrospinal fluid (CSF). CSF samples were collected from rabbits treated with alpha 2-antagonists. The aim of our study was to determine the specificity of these drugs on the central nervous noradrenergic, dopaminergic and serotonergic activity as measured by the release of corresponding monoamine metabolites. 3-Methoxy-4-hydroxyphenylethylene glycol (MHPG) and vanilmandelic acid (VMA) were used as parameters for the noradrenergic activity, whereas homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) were employed to follow the dopaminergic and serotonergic activity, respectively. For the measurement of the biogenic amine metabolites a published GCMS method has been adapted. Samples of 200 microliters CSF were processed. Following addition of deuterated internal standards and acidification, extraction was carried out with ethyl acetate. Preliminary experiments with the analysis of MHPG using diethyl ether for extraction gave rise to emulsion formation and resulted in poor recoveries for MHPG and in irreproducibility problems due to a preferential extraction of non-labelled MHPG, effects which were not observed with ethyl acetate extraction. Derivatization was done with a mixture of pentafluoropropionic anhydride/pentafluoropropanol (or hexafluoroisopropanol) in order to derivatize both hydroxyl and carboxylic acid groups. The derivatization procedure was optimized for the analysis of 5-HIAA by carrying out a second reaction step with pentafluoropropionic anhydride alone in order to complete the derivatization for the indolic NH moiety. The molecular ions of the derivatized products were selected for detection.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed2004

Quantitative determination of hexamethylene diisocyanate (HDI), 2,4-toluene diisocyanate (2,4-TDI) and 2,6-toluene diisocyanate (2,6-TDI) monomers at ppt levels in air by alkaline adduct coordination ionspray tandem mass spectrometry.

Occupational exposures to isocyanates can lead to occupational asthma. Once sensitized, some workers could react to isocyanate monomers at concentrations below 1% of the Permissible Exposure Limit of 5 ppb in air. Currently available methods are not sufficiently sensitive to adequately evaluate isocyanates present at these levels in workplace air. This article describes a novel method for isocyanate determination allowing the ultratrace quantification in workplace air of hexamethylene diisocyanate, 2,4-toluene diisocyanate and 2,6-toluene diisocyanate monomers. Sampling is performed during a complete workshift at a flow rate of 1 L min(-1) with a cassette containing a 1-(2-methoxyphenyl)piperazine-impregnated 25 mm filter. Analysis is performed using liquid chromatography hyphenated with coordination ionspray tandem mass spectrometry. The analytical method's linearity was measured for a concentration range varying from the limit of detection of 0.04-0.13 ng mL(-1), depending on the monomer, up to approximately 32 ng mL(-1) for every isocyanate monomer, all with correlation coefficients (R(2)) greater than 0.999. The analytical method's lower limit of quantification combined with an adapted sampling strategy allow the quantification of isocyanate monomers down to 0.04 ppt for an 8 h work shift when a lithium adduct is used, which is more than 300 times lower than the most sensitive method currently available. This novel method can be used to confirm the very low level of isocyanate monomers for the safe reassignment of sensitized workers and it is also useful for charting the isocyanate dispersion tail in workplace environments.

Air Pollutants, Occupational↗
PubMed1985

Adenine nucleotide and P-creatine levels in layers of frog retina as a function of dark and light adaptation.

Eight layers of frog retina were analyzed for ATP, P-creatine, ATP + ADP, and AMP under conditions of dark, 2 sec, 2 min, and in the case of AMP, 2 hr of light adaptation. Samples of each layer, usually ranging between 5 and 50 ng, were dissected from lyophilized frozen sections. After brief light exposure, ATP dropped while ADP rose sharply in the pigment epithelium, outer segments, and inner segments; ADP was too low to be measured accurately in the inner retina. The profile of ATP, P-creatine, and ATP + ADP concentrations showed peaks in the inner nuclear and ganglion layers. AMP, by contrast, was highest in the two plexiform layers. Levels in the inner retina dropped after only 2 sec of light but rose after 2 hr to levels that were higher than dark values in all retinal layers. AMP was often characterized by a non-uniform distribution: adjacent areas of a layer agreed very closely in value to each other but could vary several-fold from a different section of the same frog or from another frog exposed to the same conditions. This distribution produced clusters of values, particularly prominent in dark-adapted animals, something not observed with the other metabolites measured. The peaks of AMP in the plexiform layers suggest that AMP may be a by-product of dopamine-stimulated adenylate cyclase which also has peaks in these same layers.

Adenine Nucleotides↗
PubMed2004

Comparative analysis of transcriptional profiles between two apoptotic pathways of light-induced retinal degeneration.

Light exposure can exacerbate the condition of a variety of human retinal diseases by increasing the rate of photoreceptor cell death. How light negatively affects photoreceptor cell survival is not yet fully understood. Previous studies involving light damage models have revealed two independent apoptotic pathways: low levels of light induce retinal degeneration in the arrestin -/- mouse via constitutive activation of the phototransduction cascade, whereas strong light exposure to the retina, such as in an albino eye, elicits photoreceptor cell death via activator protein (AP-1) induction. In order to better understand the initial gene expression changes underlying light damage, dark-reared arrestin -/- and albino BALB/c mice were exposed to constant white light (2000 lux), and their retinal morphology was assessed as a function of time. The expression profiles of retinal transcripts were then compared between dark-adapted and light-exposed arrestin -/-, pigmented wild-type and BALB/c mice at a time point when morphological changes were minimal. As expected, the dark-adapted samples showed little difference in expression pattern between the three genotypes. Among the genes differentially regulated by light in BALB/c, but not arrestin -/- retinas, were c-fos and other stress-induced early response genes. In both mouse models, a marked increase in expression of the bZIP family of transcription factors was observed. Our results show a select group of unique and overlapping sets of genes induced by light in the two mouse models. These expression changes may constitute the underlying initiating events leading to the two distinct mechanisms of light damage.

Animals↗
PubMed1991

Unusual low reactivity of the water oxidase in redox state S3 toward exogenous reductants. Analysis of the NH2OH- and NH2NH2-induced modifications of flash-induced oxygen evolution in isolated spinach thylakoids.

The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].

Hydrazines↗
PubMed1990

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin.

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.

Bacteriorhodopsins↗
PubMed2004

The effects of family resources, coping, and strains on family adjustment 18 to 24 months after the NICU experience.

PURPOSE: To examine the relationship of family coping, resources, and strains on family adjustment over time following the NICU experience. DESIGN: Longitudinal, correlational study based on the Resiliency Model of Family Stress, Adjustment and Adaptation. SAMPLE: Data were collected, through mailed questionnaires, from 71 couples, 18 to 24 months following the birth of their infant. The data are a follow-up from a previous study conducted at the time of the infant's birth. MAIN OUTCOME VARIABLES: The dependent variable in the study was family adjustment, measured by the McMaster Family Assessment Device. The independent variables were family resources, as measured by the Family Inventory of Resource Management; family coping, as measured by the Family Crisis Oriented Evaluation Scales; family strain, as measured by the Family Inventory of Life Events and Changes; and parent gender, family system (first-time parent or not), and the child's health, as measured by the Demographic Information Questionnaire. RESULTS: Family adjustment improved over time for mothers but decreased for fathers. Fathers of infants with ongoing health problems reported significantly poorer family adjustment. Family resources were related to family adjustment and decreased over time for both parents. Families used more coping mechanisms and different coping patterns over time.

Adaptation, Psychological↗
PubMed2001

Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae.

An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.

Biomass↗
PubMed1980

The orientation of the magnetic axes of the membrane-bound iron-sulfur clusters of spinach chloroplasts.

Spinach chloroplast membranes were oriented onto mylar sheets by partial dehydration, and the orientation of the magnetic axes of membrane-bound paramagnetic clusters determined by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the reduced Rieske iron-sulfur cluster signal is of orthorhombic symmetry oriented with th gy = 1.90 axis orthogonal to the membrane plane and with the gz = 2.03 axis in the membrane plane; the gx-axis is undetectable, presumably due to its broadness. If the Rieske center is a two-iron iron-sulfur cluster, we conclude that the iron-iron axis lies in the plane of the membrane. Illumination reduces the two bound chloroplast iron-sulfur proteins known as Clusters A and B. Center A is oriented such that gx = 1.86 and gy = 1.94 lie at an angle of about 40, and gz = 2.05 is at approximately 25, to the membrane plane. There are two possible orientations of Cluster B depending on the set of g-values assigned to this cluster. For one set of g-values, gz = 2.04 and gx = 1.89 are oriented in the plane of the membrane while gy = 1.92 is orthogonal to the plane. Alternatively, gz = 2.07 and gy = 1.94 are oriented approximately 50 and 40 to the membrane plane respectively, and gx = 1.80 is in the plane of the membrane. An additional light-induced signal at g = 2.15 oriented orthogonal to the plane is currently unexplained, as are other membrane perpendicular signals seen at g = 2.3 and g = 1.73 in dark-adapted samples.

Cell Membrane↗
PubMed1984

Interaction of linolenic acid with bound quinone molecules in Photosystem II. Time-resolved optical and electron spin resonance studies.

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.

Chloroplasts↗
PubMed1992

Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942.

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40 degrees C in wild-type thylakoids to about 30 degrees C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak termperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of QB. (e) Mutations at the QB site not only influence the binding of QB and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S2 and S3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the QB site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and QB, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect QB or DCMU binding in Synechococcus PCC7942.

Atrazine↗
PubMed2005

Absorption and emission spectroscopic characterisation of combined wildtype LOV1-LOV2 domain of phot from Chlamydomonas reinhardtii.

An absorption and emission spectroscopic characterisation of the combined wild-type LOV1-LOV2 domain string (abbreviated LOV1/2) of phot from the green alga Chlamydomonas reinhardtii is carried out at pH 8. A LOV1/2-MBP fusion protein (MBP=maltose binding protein) and LOV1/2 with a His-tag at the C-terminus (LOV1/2-His) expressed in an Escherichia coli strain are investigated. Blue-light photo-excitation generates a non-fluorescent intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm). The photo-cycle dynamics is studied by dark-state absorption and fluorescence measurement, by following the temporal absorption and emission changes under blue and violet light exposure, and by measuring the temporal absorption and fluorescence recovery after light exposure. The fluorescence quantum yield, phi(F), of the dark adapted samples is phi(F)(LOV1/2-His) approximately 0.15 and phi(F)(LOV1/2-MBP) approximately 0.17. A bi-exponential absorption recovery after light exposure with a fast (in the several 10-s range) and a slow component (in the near 10-min range) are resolved. The quantum yield of photo-adduct formation, phi(Ad), is extracted from excitation intensity dependent absorption measurements. It decreases somewhat with rising excitation intensity. The behaviour of the combined wildtype LOV1-LOV2 double domains is compared with the behaviour of the separate LOV1 and LOV2 domains.

Abstracting and Indexing↗
PubMed1995

Photocycle of halorhodopsin from Halobacterium salinarium.

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.

Bacteriorhodopsins↗
PubMed1998

On the origin of the '35-mus kinetics' of P680(+.) reduction in photosystem II with an intact water oxidising complex.

The origin of the '35-micros kinetics' of P680(+.) reduction in photosystem II (PS II) with an intact water oxidising complex has been analysed by comparative measurements of laser flash induced changes of the 830-nm absorption and the relative quantum yield of chlorophyll (Chl) fluorescence. The latter parameter was monitored at a time resolution of 500 ns by using newly developed home built equipment [Reifarth, F., Christen, G. and Renger, G. (1997) Photosynth. Res. 51, 231-2421. It was found that: (i) the amplitudes of the unresolved ns-kinetics of both 830-nm absorption changes and the rise of fluorescence yield exhibit virtually the same period four oscillation pattern when dark adapted samples are excited with a train of saturating laser flashes; (ii) the corresponding oscillation patterns of the normalised extent of the 35-micros kinetics under identical excitation conditions are strikingly different with maxima after the 3rd and 5th flash for the 830-nm absorption changes vs. pronounced maxima after the 4th and 8th flash for the rise of the fluorescence yield. The period four oscillations unambiguously show that the '35-micros kinetics' of P680(+.) reduction are characteristic for reactions in PS II entities with an intact water oxidising complex. However, the disparity of the oscillation patterns of (ii) indicates that in contrast to the ns components of P680(+.) reduction the 35-micros kinetics do not reflect exclusively an electron transfer from Y(Z) to P680(+.). It is inferred that a more complex reaction takes place which comprises at least two processes: (a) P680(+.) reduction by Y(Z) and (b) coupled and/or competing reaction(s) which give rise to additional changes of the chlorophyll fluorescence yield.

Chlorophyll↗
PubMed1997

Stochastic optimization algorithms of a Bayesian design criterion for Bayesian parameter estimation of nonlinear regression models: application in pharmacokinetics.

This article proposes three stochastic algorithms to optimize a Bayesian design criterion for Bayesian estimation of the parameters of nonlinear regression models; this criterion is the information expected from an experiment. The first algorithm is based on a stochastic version of the simplex with an adaptive sampling procedure. The others are stochastic approximation algorithms: the Kiefer-Wolfowitz and the pseudogradient algorithms. We first present the information criterion and the optimization algorithms. The efficiency of each algorithm for optimizing this Bayesian design criterion is then assessed by a simulation study for a nonlinear model assuming a discrete prior distribution. An application for designing an experiment to estimate the kinetics of radioiodine thyroid uptake is then proposed.

Algorithms↗
PubMed2003

Estimation following group-sequential response-adaptive clinical trials.

A sequential clinical trial model is considered in which two treatments with immediate normally distributed responses are to be compared. The class of one-sided group-sequential tests with response-adaptive sampling developed by Jennison and Turnbull is used to investigate which of the treatments has the larger mean response. The power function for this class of tests is the same as that under nonadaptive sampling, and significant decreases in the inferior treatment number can be achieved with only minor increases in the average total sample number. Two inferential methods are considered following the design. Approximate confidence intervals for the treatment mean difference and the individual means are constructed using the pivotal method of Woodroofe, and an approximation to the bias of the maximum likelihood estimator of the treatment mean difference is studied based on the work of Whitehead. Simulation is used to assess the accuracy of both methods for various stopping boundaries and numbers of interim analyses.

Clinical Trials as Topic↗
PubMed2000

NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat.

The metabolic profiles of three wild mammals that vary in their trophic strategies, the herbivorous bank vole (Clethrionomys glareolus), the granivorous wood mouse (Apodemus sylvaticus), and the insectivorous white-toothed shrew (Crocidura suaveolens), were compared with that of a widely used strain of laboratory rat (Sprague Dawley). In conjunction with NMR spectroscopic investigations into the urine and blood plasma composition for these mammals, high resolution magic angle spinning (HRMAS) 1H-nuclear magnetic resonance (NMR) spectroscopy was applied to investigate the composition of intact kidney samples. Adaptation to natural diet affects both renal metabolism and urinary profiles, and while these techniques have been used to study the metabolism of the laboratory rat little is known about wild small mammals. The species were readily separated by their urinary profiles using either crude metabolite ratios or statistical pattern recognition. Bank vole urine contained higher concentrations of aromatic amino acids compared with the other small mammals, while the laboratory rats produced relatively more hippurate. HRMAS 1H-NMR demonstrated striking differences in both lipid concentration and composition between the wild mammals and Sprague Dawley rats. Bank voles contained high concentrations of the aromatic amino acids phenylalanine, tyrosine and tryptophan in all tissue and biofluids studied. This study demonstrates the analytical power of combined NMR techniques for the study of inter-species metabolism and further demonstrates that metabolic data acquired on laboratory animals cannot be extended to wild species.

Animals↗
PubMed1998

High-throughput solid-phase extraction for the determination of cimetidine in human plasma.

For the implementation and validation of an automated 'high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C18 sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.

Chromatography, High Pressure Liquid↗