Deproteinization with ZnSO4–Ba(OH)2 reduces the photodegradation of montelukast during plasma sample preparation for HPLC analysis

Montelukast (MKT), a leukotriene receptor antagonist, degrades when it is exposed to light. The analysis of MKT content in blood plasma by high-pressure liquid chromatography requires several sample preparation steps including deproteinization. This study aimed to evaluate MKT photodegradation in blood plasma samples and optimize a deproteinization method to reduce MKT photodegradation, and thereby improve analytical quality. We evaluated the stability of MKT in water and plasma in real time using high-pressure liquid chromatography and optimized a sample deproteinization procedure by comparing the effectiveness of several deproteinization methods. When exposed to light, MKT photodegraded quickly. Although MKT photodegradation was slightly slower than that in water, a half portion (55%) of the MKT in plasma degraded within 2 h when exposed to light. The rate of MKT photodegradation was dramatically reduced by sample deproteinization using ZnSO₄–Ba(OH)₂, but it was accelerated by deproteinization through precipitation using methanol or acetonitrile. These results suggest that precautions should be taken when preparing plasma samples for the analysis of MKT, and that deproteinization of such samples using ZnSO₄–Ba(OH)₂ can reduce the risk of analytical error arising from MKT photodegradation.     

MEDIATION OF LIGHT-INDUCED CHANGES IN PINEAL RECEPTOR AND SUPPORTING CELL NUCLEI AND NUCLEOLI IN STEELHEAD TROUT (SALMO GAIRDNERI)

Abstract— Mediation of the chronic effects of differences in environmental illumination on pineal photoreceptor and adjacent supporting cells was studied in two experiments using larval trout. Experiment 1 had 7 groups exposed to controlled lighting conditions for 14 months: (1) constant darkness (DD); (2) natural photocycles (LD) followed by constant light (LL); (3) natural photocycles (LD); (4) LD fluorescent light; (5) LD blue (400–525nm) light; (6) LD green (500–612nm) light; and (7) LD red (600–750 nm) light. In Experiment II the fish were exposed to LL for 27–28 days, following 10 months in DD and one of five surgical manipulations: (1) pineal masked; (2) pineal sham-masked; (3) blinded; (4) blinded + pineal masked; (5) blinded + pineal sham masked.
Nuclei and nucleoli of pineal receptor and supporting cells were largest in fish raised in DD. LL or LD cycles, and light of either narrow or broad spectral range, were not notably different in their chronic effects on receptor cell nuclei and nucleoli. However, supporting cell nuclei and nucleoli were smallest in LL. Blinding and pineal masking demonstrated that: (1) pineal receptor cell nuclei and nucleoli were affected by chronic incident light in the pineal region but not by photic or visual influences via the lateral eyes; and conversely, (2) pineal supporting cell nuclei and nucleoli were affected by chronic photic input only via the lateral eyes. The results suggest the possible presence of a cellular comparator system in which photic inputs from lateral eyes and pineal affect separately and respectively the adjacent supporting and receptor cells of the pineal organ.     

Photochemical decomposition of lomefloxacin in vitro and in vivo

To obtain an idea of the photostability of Lomefloxacin (Lom) under in vivo conditions the compound was exposed to UV-A (310-360 nm) in PBS buffer pH 7.4. Exposure of 10 microg/ml of Lom in PBS pH 7.4 led to more than 50% decomposition within 10 min. Loss of the fluorine atom at C-8 and partial breakdown of the piperazine ring occurred. The only two photoproducts formed under these conditions were AEA, 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminoethyl-amino)-4-oxo-3-quinolinecarboxylic acid, and APA, 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminopropyl-amino)-4-oxo-3-quinolinecarboxylic acid. When Lom was exposed in whole blood in vitro, the same photochemical decomposition was observed in the plasma as in PBS buffer: APA and AEA were the only products. During UV-A exposure, Lom was shown to be taken up by the leukocytes. This process appeared to be less rapid during UV-A exposure than in the dark. As soon as UV-A exposure commenced, AEA and APA were found. As in the plasma, the total amount of Lom and the two photoproducts in the leukocytes was not significantly different from the amount of Lom found in unexposed cells at the same time point. The erythrocytes did not take up Lom, but exposure of whole blood to Lom and UV-A under the above conditions led to more than 7% haemolysis. Treatment of rats with a combination of Lom and UV-A demonstrated photodecomposition of Lom in vivo. In urine produced during exposure and by the irradiated rats during the twilight period after exposure, a considerable amount of AEA and APA was found. The blood plasma from rats exposed simultaneously to UV-A and Lom proved to contain AEA and APA and, in the leukocytes, APA. This was not the case with animals kept in twilight.     

Quantitation of N epsilon-(dichloroacetyl)-L-lysine in proteins after perchloroethene exposure by gas chromatography-mass spectrometry using chemical ionization and negative ion detection followingimmunoaffinity chromatography.

An antibody specific to N epsilon-(dichloroacetyl)-L-lysine (DCA-Lys) was immobilized to immunoaffinity columns for the use in selective enrichment of dichloroacetylated proteins. These result from the reaction with dichlorothioketene the beta-lyase cleavage product of the perchloroethene metabolite S-(trichlorovinyl)-L-cysteine. Dichloroacetylated proteins from rat kidney mitochondria, rat plasma and human blood plasma were isolated after exposure to 40 ppm tetrachloroethene (PER) for 6 h. After acid hydrolysis of the protein fraction, DCA-Lys was derivatized with 1,3-dichloro-1,1,3,3-tetrafluoroacetone using N epsilon-(trifluoroacetyl)-L-lysine as internal standard. Recovery of dichloroacetylated reference proteins from immunoaffinity columns was about 73%. Samples were analyzed by GC-MS with chemical ionization and negative ion (NCI) detection showing DCA-Lys in proteins with 2.26 (+/- 0.02) pmol/mg protein in male rat kidney mitochondria and 1.92 (+/- 0.05) pmol/mg total mitochondrial protein in female rats. In rat plasma 0.47 (+/- 0.006) pmol DCA-Lys/mg protein in male and 0.34 (+/- 0.02) in female animals were found. DCA-Lys could not be detected in blood plasma of human volunteers exposed to PER with a detection limit of 20 fmol for the DCA-Lys derivative 2,2-bis(chlorodifluoromethyl)-4-(1-dichloroacetamido)-butyl- 1,3-oxazolidine-5-one. Immunoaffinity chromatography with specific antibodies provides a powerful tool for the enrichment of minor quantities of dichloroacetyled proteins in biological samples for GC-NCI-MS analysis of the modified amino acid lysine having broad utility in the biomonitoring of PER exposure.     

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

Isolation of cell-free DNA from plasma by chromatography on short monolithic columns and quantification of non-apoptotic fragments by real-time polymerase chain reaction

Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.     

EFFECT OF PHOTIC STIMULATION ON THE HUMAN MENSTRUAL CYCLE

To test the hypothesis that artificial light can be used to regularize the human menstrual cycle, 16 women with histories of menstrual irregularity and/or abnormally long cycles were studied. It was assumed that a 29-day cycle is the "normal" length. Thus, light exposure was begun on the evening of day 14, with the expectation that ovulation would be triggered by day 15 and menstruation on day 30. The light regimen was continued for 2-3 nights more. Temperature graphs indicated all subjects were ovulating. 41 control and 41 matching experimental cycle lengths were obtained. The experimental cycle lengths were less variable than the control lengths and showed a sharp peak at 29 days. In the 11 subjects exposed to light for more than 1 cycle, 9 showed a decrease in the range of cycle length (3.3% level of significance difference between control and experimental groups in terms of cycle length). It is concluded that these data provide evidence that photic stimulation can regularize menstrual cycle length and thus influence the time of ovulation, thereby facilitating use of the rhythm method of birth control. Further studies involving larger samples and more sophisticated measurements of ovulation in relation to photic stimulation are recommended.     

PHOTOSENSITIVITY IN THE SKIN OF THE LIZARD, Anolis carolinensis

Skin of the lizard, Anolis carolinensis, is remarkably sensitive to light. Illuminated in vitro with visible light from a tungsten source (110 W (m-2)), skin changes from brilliant green to dark brown (50% reduction in reflectance) within 2-4 min as a result of dispersion of melanin from a perinuclear position within dermal melanophores into superficial dendritic processes. Reversal of the process, reaggregation of pigment, will occur within 2.0 min upon return to darkness. This photic response can be initiated with light levels as low as 5.0 W m(-2) and is maximized by light levels only 5% that of midwinter sunshine. Pigment dispersion in response to both melanocyte simulating hormone and to light is inhibited by cytochalasin-B, indicating that microfilaments may be the motor element for pigment movement in that direction. Colchicine, but not cytochalasin-B, totally blocks pigment reaggregation following melanocyte stimulating hormone exposure and partially blocks it in the dark phase of the photic response. The results of this study are consistent with a model for pigment movement in A. carolinensis that provides microfilaments for pigment dispersion and microtubule involvement in both dispersion and aggregation. Finally, because it is readily visible, easily quantified, rapid and reversible, photic response in the skin of A. carolinensis is recommended as a valuable model system for the study of saltatory movement of organelles within cells.     

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). Part III: Lymphocytes

The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a T-cell-specific mitogen, concanavalin A (ConA), or a B-cell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVB-exposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine gamma-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyte-related functions in fish.     

Arsenobetaine-decomposing Ability of Marine Microorganisms Occurring in Particles Collected at Depths of 1100 and 3500 Meters

The arsenobetaine-decomposing ability of microorganisms occurring in sinking particles, which play a main role in the vertical transport of organic substances produced in the photic zone, was investigated. The microorganisms in particles collected in the deep sea, 1100 and 3500 m in depth, clearly showed decomposing ability. With the particles from 1100 m, the degradation products were the same as those produced by microorganisms occurring in sources in the photic zone, i.e. trimethylarsine oxide (TMAO), dimethylarsinic acid (DMA) and inorganic arsenic(V). At 3500 m, the degradation activity was diminished, smalls amount of DMA and TMAO being produced. These results suggest that arsenobetaine contained in the animals starts to degrade immediately after the death of the animals and their transformation to particles. The degradation of arsenobetaine to inorganic arsenic in our tentative arsenic cycle in marine ecosystems (inorganic arsenic to inorganic arsenic via the biosynthesis of arsenobetaine) may apply to the deep sea as well as to the photic zone. © 1997 by John Wiley & Sons, Ltd.     

急性镉中毒大鼠致死时重要器官镉的分布

分别通过静脉和呼吸道急性镉染毒，探讨了急性镉中毒大鼠致死时，血液，心、肝、肾、脑、肺的镉分布。８０只Ｗｉｓｔａｒ大鼠分为对照组和染毒组，氯化镉进行急性染毒，在心肌收缩功能降低为染毒前的５０％及心跳停止时，取器官标本，用原子吸收分光光度计测定镉含量。实验结果提示，急性镉中毒时，血液、心脏的镉含量早期升高缓慢，后期升高较快，肝脏镉含量呈线性快速升高；而肾脏镉含量经呼吸道染毒时与肝脏类似，经静脉染毒时，     

长期低浓度铅暴露对人体微量元素的影响

测定了１５名长时间从事低剂量铅接触的女工血铅、铜、锌、铁、锰及铜锌超氧化物歧化酶活性、脂质过氧化产物二级降解物丙二醛含量,并以同年龄组同地区非铅暴露人群作对照。结果显示：暴露组的血铅、铜、ＭＤＡ含量高于对照组;而血锌Ｃｕ－Ｚｎ－ＳＯＤ活性低于对照组;血Ｍｎ、Ｆｅ两组无明显差异。     

免疫吸附治疗用ProteinA切流膜色谱柱的研究

切流膜色谱柱是免疫吸附治疗中实现全血灌流的一种新的模式。实验研究表明,切流膜色谱柱的结构和血液流动形式对血细胞的损害很小。分别考察了水、血浆、血液等不同流体流速与切流膜色谱柱柱压降之间的关系,柱压降随着流体流速和粘度的升高而增高,血液流速为120mL／min时,柱压降达到93kPa;考察了ProteinA切流膜色谱柱对人血浆中免疫球蛋白IgG的吸附能力,切流膜色谱柱ProteinA健合量为139mg（6mgProteinA／g干介质）,血浆在柱中循环1h可吸附IgG553mg（23．8mgIgG／g平介质）,而血液在柱中循环1h,可吸附IgG499.4mm（21.5msIgG／g卡介质）。     

Comparative effects of UVA and UVB irradiation on the immune system of fish

Aquatic organisms can be harmed by the current levels of solar ultraviolet radiation. We have recently shown that exposure of fish to UVB irradiation alters the functioning of the fish immune system, but the effects of UVA radiation are unknown. The present study continues this work by characterizing UVA irradiation-induced immunological changes in fish. Roach, a cyprinid fish, were exposed to a single dose of either UVA (3.6 J/cm2) or UVB (0.5 J/cm2) irradiation. Both irradiations suppressed transiently mitogen-stimulated proliferation of blood lymphocytes. UVA, but not UVB, decreased hematocrit, plasma protein, and plasma immunoglobulin levels and increased the proportions of blood cells classified as unidentified leukocytes, possibly consisting of UVA-damaged lymphocytes. UVB, but not UVA, altered the functioning of head kidney and blood phagocytes, induced granulocytosis and lymphocytopenia in the blood and increased plasma cortisol concentration. These results imply that both UVA and UVB are potent modulators of the immune defence of fish.     

Response of Linear,Branched or Crosslinked Polyethylene Structures on the Attack of Oxygen Plasma

Linear, branched and crosslinked polyethylenes (PE) were exposed to the low-pressure oxygen plasma for 2–120 s. In the following the samples were washed with solvents to remove low-molecular weight oxidized material and to excavate the subjacent polymer structure for microscopic characterization. X-ray photoelectron spectroscopy (XPS) measurements provided information about changes in elemental composition and chemical structure of PE after plasma exposure and washing. The calculation of the concentration of tertiary C atoms using XPS data was a measure of branches and crosslinking in the polymer before and after exposure to oxygen plasma. Linear PE was most sensitive towards oxygen plasma and showed the highest concentration in tertiary C atoms after plasma exposure. On the other hand branched PE types, which possess originally more tertiary carbon atoms, have lost two-third of them after 2 s oxygen plasma exposure. Branched PE show also topological changes at their surface as detected by atomic force microscopy. Differential scanning calorimetry measurements confirmed strong changes in crystallinity and molecular orientation of linear PE already after 120 s exposure to the oxygen plasma interpreted as amorphization. These effects should be interpreted as result of crosslinking caused by the recombination of dangling bond sites.     

Penetration of solar radiation into the waters of Messina Strait (Italy)

The optical properties of the waters of five different stations, three located in the Messina Strait and two near the Strait (open sea), were analysed. Direct spectral measurements of the downward solar irradiance (290 - 800 nm) at different depths (0.5 m, 7 m, 10 m, 13 m, 20 m) were made using a cosine sensor connected to a spectroradiometer. Water samples were collected in the surface layer and their absorption spectra were analysed. The natural fluorescence profiles, along the water column, were determined using a fluorometer (SBE 911plus - Sea Teach). The spectral attenuation coefficient (K(lambda)), the variation of K(lambda) in different wavelength ranges (deltaK(deltalambda)), the wavelength corresponding to minimum value of K(lambda), the spectral depths of penetration of both 1% and 10% of the sub-surface irradiance values (P(lambda)), the depths of 1% of penetration of UVB, UVA and PAR, the depth ranges of the maxim concentration of Chl a and superficial CDOM were measured at each station. The maximum solar UVB penetration was about 65% of the photic zone and the maximum UVA penetration was nearly 100% (data of the Ionic sea station). Thus, a large part of the photic zone was exposed to UV radiation sufficient to cause a possible reduction in the photosynthetic activity of phytoplankton. The spectral penetration of solar radiation, especially UVB radiation, was significantly different in the three stations of the Strait with respect to the two stations studied in the open sea. This shows that variations in the spectral attenuation along the water column can be used as an indicator of properties of the water body.     

Comparison of two parameter choice methods for the extraction of depth profiles by means of a regularized multipoint profile model from ARXPS data obtained on a three‐component system

Polystyrene films were exposed to nitrogen plasmas for periods up to 8 min. Angle‐resolved X‐ray photoelectron spectroscopy measurements revealed the presence of oxygen and nitrogen in the surface due to the plasma treatment. The depth profiles of these adatoms were determined by fitting a regularized multipoint linear segment model to the data. A regularization parameter chosen such that the chi‐square statistic of the fit to the data was equal to the number of independent data points gave a more intuitive result than a parameter chosen according to the L‐curve criterion. Although the shape of the nitrogen depth profile was observed to vary as a function of the plasma duration, the oxygen depth profiles were nearly identical. Copyright © 2009 John Wiley & Sons, Ltd.     

A microfluidic device for continuous, real time blood plasma separation

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach-Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device's simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.     

Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study

A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.     

Fatty acid profiling of raw human plasma and whole blood using direct thermal desorption combined with gas chromatography-mass spectrometry

Gas chromatography (GC) has in recent times become an important tool for the fatty acid profiling of human blood and plasma. An at-line procedure used in the fatty acid profiling of whole/intact aquatic micro-organisms without any sample preparation was adapted for this work. A direct thermal desorption (DTD) interface was used to profile the fatty acid composition of human plasma and whole human blood of eight volunteers in a procedure omitting the usual lipid extraction steps that precede sample methylation in the traditional (off-line) protocols. Trimethylsulfonium hydroxide (TMSH) was used as reagent for thermally assisted methylation. In a fully automated manner, the liner of the GC injector is used as a sample-and-reaction container with the aid of the DTD interface. The fatty acid methyl ester (FAME) profiles obtained using this novel approach, were very identical to those obtained when the traditional off-line protocol was applied. FAME yields obtained in the at-line DTD method were found to be very similar for saturated fatty acids, but significantly higher for polyunsaturated fatty acids compared to off-line yields. As a result of the contribution of circulating cell membranes in blood, substantial differences were observed when the amount of FAMEs obtained in whole human blood and human plasma samples were compared after their analysis. Thanks to the fully automated operation of this novel procedure, large series of analyses can easily be performed.