Isotopic analysis of Fe in human red blood cells by multiple collector-ICP-mass spectrometry.

Precise 56Fe/54Fe and 57Fe/54Fe isotopic ratios on human red blood cell (RBC) samples have been measured using multiple collector-ICP-mass spectrometry (MC-ICPMS). The mass spectrometric interferences on Fe isotopes (e.g., 56ArO+ and 57ArOH+) were successfully minimized by a dry plasma condition achieved by a desolvating nebulizer sample-introduction technique. In order to eliminate possible variations in the measured isotopic ratios due to non-mass spectrometric interferences, Fe was separated from remaining organic compounds and major co-existing elements using an ion chromatographic technique. The resulting precisions of the 56Fe/54Fe and 57Fe/54Fe ratio measurements were 0.12 per thousand and 0.20 per thousand, respectively, which were high enough to detect the isotopic variation of Fe in nature. For an interlaboratory comparison, all of the Fe isotopic ratio data were normalized by the ratios for the IRMM-014 international isotopic standard. A series of 12 RBC samples were collected from one person through monthly-based sampling over a period of one year. These were analyzed to test possible seasonal changes in the 56Fe/54Fe and 57Fe/54Fe ratios. Moreover, in order to test possible variations in the 56Fe/54Fe and 57Fe/54Fe ratios among different people, RBC samples were collected from five volunteers (four males and one female). The 56Fe/54Fe and 57Fe/54Fe ratios for a series of 12 RBC samples collected over a one-year period show 3.06 per thousand and 4.51 per thousand lower than the values of IRMM-014, and no significant seasonal change could be found in the ratios. The lack in seasonal changes in the Fe isotopic ratios could be explained by a small contribution of the daily net-intake of Fe (1 - 2 mg/day) onto the total amount of Fe in the human body (2 - 4 g). The 56Fe/54Fe and 57Fe/54Fe ratios for RBC samples collected from four male samples did not vary measurably, whereas the Fe isotopic ratios for a female RBC were 0.3 per thousand/amu heavier than the mean value of four male samples. This difference in Fe isotopes among the individuals can be the result of a difference in uptake efficiency of the Fe through a dietary process from the digestive tract. The data obtained here demonstrate that the isotopic ratios of trace metals can provide new information about metabolic efficiencies of the metallic elements.     

High-throughput biophysical measurement of human red blood cells

This paper reports a microfluidic system for biophysical characterization of red blood cells (RBCs) at a speed of 100-150 cells s(-1). Electrical impedance measurement is made when single RBCs flow through a constriction channel that is marginally smaller than RBCs' diameters. The multiple parameters quantified as mechanical and electrical signatures of each RBC include transit time, impedance amplitude ratio, and impedance phase increase. Histograms, compiled from 84,073 adult RBCs (from 5 adult blood samples) and 82,253 neonatal RBCs (from 5 newborn blood samples), reveal different biophysical properties across samples and between the adult and neonatal RBC populations. In comparison with previously reported microfluidic devices for single RBC biophysical measurement, this system has a higher throughput, higher signal to noise ratio, and the capability of performing multi-parameter measurements.     

Influence of PAMAM dendrimers on human red blood cells

Polyamidoamine (PAMAM) dendrimers with different concentrations (1 nM-1 mM) (generations 2, 3, and 4) impact on human red blood cell morphology, and membrane integrity is studied. Erythrocyte shape changes from biconcave to echinocytic in dendrimers. Cell aggregation occurs. Polymers cause also concentration- and generation-dependent haemolysis.     

High-performance liquid chromatographic determination of cyanide in human red blood cells by pre-column fluorescence derivatization.

A method for the determination of cyanide in human red cells has been developed. Cyanide was extracted from red cells by adding water and methanol, and then derivatized with 2,3-naphthalene-dialdehyde and taurine to give a fluorescent product, which was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. The recovery of cyanide from red cells was ca. 83%, and the limit of detection was 100 pmol/ml. The mean concentrations of red cell cyanide from ten smokers and from ten non-smokers were 705 and 466 pmol/ml, respectively. The method was also applicable to whole blood.     

Analysis of enzymopathies in the human red blood cells by constraint-based stoichiometric modeling approaches

The human red blood cell (RBC) metabolism is investigated by calculating steady state fluxes using constraint-based stoichiometric modeling approaches. For the normal RBC metabolism, flux balance analysis (FBA) is performed via optimization of various alternative objective functions, and the maximization of production of ATP and NADPH is found to be the primary objective of the RBC metabolism. FBA and two novel approaches, minimization of metabolic adjustment (MOMA) and regulatory on-off minimization (ROOM), which can describe the behavior of the metabolic networks in case of enzymopathies, are applied to observe the relative changes in the flux distribution of the deficient network. The deficiencies in several enzymes in RBC metabolism are investigated and the flux distributions are compared with the non-deficient FBA distribution to elucidate the metabolic changes in response to enzymopathies. It is found that the metabolism is mostly affected by the glucose-6-phosphate dehydrogenase (G6PDH) and phosphoglycerate kinase (PGK) enzymopathies, whereas the effects of the deficiency in DPGM on the metabolism are negligible. These stoichiometric modeling results are found to be in accordance with the experimental findings in the literature related to metabolic behavior of the human red blood cells, showing that human RBC metabolism can be modeled stoichiometrically.     

On the temperature dependence of the dielectric membrane properties of human red blood cells

Electrorotation (ER) spectra of human red blood cells (HRBCs) have been recorded in the frequency range from 10 kHz to 250 MHz in a 4-electrode microchip chamber. The cells were suspended at conductivities in the range from 0.02 to 3.00 S/m (corresponding to an ionic strength range from 1.6 to 343 mM) at temperatures between 10 degrees C and 35 degrees C. Generally, the characteristic frequencies as well as the rotation speeds of the first (membrane-dispersion) and second ER peaks increased with temperature. The rotation speed increase was largely correlated to the temperature dependence of the medium viscosity. Standard temperature dependencies were assumed for the conductivities and permittivities of cytoplasm, membrane, and external solution to explain the frequency shifts, starting from the cell parameters of Gimsa et al. [Gimsa et al., 1996, Biophys. J. 71: 495-506.]. The membrane capacitance was assumed to be temperature independent, based on the permittivity of alkyl-chains. Under these assumptions, the spectra could be well fitted only in a narrow temperature range around 20 degrees C. The temperature dependence of the first characteristic frequency was much stronger than predicted. In addition, around 15 degrees C, an anomalously high rotation speed was observed for the first peak at low external conductivities. Interestingly, this finding corresponds to the change in the chloride transport rate described by Brahm [Brahm, 1977, J. Gen. Physiol. 70: 283-306.].     

Spectrophotometric determination of manganese in human plasma and red cells with benzohydroxamic acid

A spectrophotometric method for the determination of manganese in human plasma and red cells with benzohydroxamic acid is presented, Interfering ions are removed by an anion exchange method, using Dowex-1, 8X resin. The manganese concentration found for plasma and red cells was about 0.1 and 0.2 p.p.m , respectively.     

Changes in the intracellular Ca2+ content in human red blood cells in the presence of glycerol

Changes of the intracellular Ca2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze-thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca2+ pump. After inhibiting the Ca2+ pump using o-vanadate, the Ca2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze-thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca2+ content. Also in this case, the Ca2+ pump seemed to play a major role.     

L-ergothioneine level in red blood cells of healthy human males in the Western province of Saudi Arabia

Ergothioneine is widely distributed in biological systems, particularly in red blood cells of animals. However, it's functional role in human body is not well understood. In order to investigate the biochemical effect of L-ergothioneine, its concentration changes in human blood with respect to ages in healthy individuals was first investigated. L-ergothioneine concentrations in the blood of Saudi males from western province at different stages of life were measured by the procedure of Carlsson et al., 1974. At early stages of life (1-10 years), the concentrations of LER is 1.5-2.0 mg/100 ml. It increases gradually at the age of 11-18 years where it reaches the maximum value of 3.7 mg/100 ml. Then, it declines gradually to 3.0-2.3 mg/ 100 ml during the period of 19-50 years. An increase in the level of LER (2.8 mg/100 ml) was seen at the age of 51+.     

Photoinduced elimination of senescent microglia cells in vivo by chiral gold nanoparticles

Parkinson''s disease (PD) is an age-related neurodegenerative disease, and the removal of senescent cells has been proved to be beneficial for improving age-associated pathologies in neurodegeneration disease. In this study, chiral gold nanoparticles (NPs) with different helical directions were synthesized to selectively induce the apoptosis of senescent cells under light illumination. By modifying anti-B2MG and anti-DCR2 antibodies, senescent microglia cells could be cleared by chiral NPs without damaging the activities of normal cells under illumination. Notably, l-P⁺ NPs exhibited about a 2-fold higher elimination efficiency than d-P⁻ NPs for senescent microglia cells. Mechanistic studies revealed that the clearance of senescent cells was mediated by the activation of the Fas signaling pathway. The in vivo injection of chiral NPs successfully confirmed that the elimination of senescent microglia cells in the brain could further alleviate the symptoms of PD mice in which the alpha-synuclein (α-syn) in cerebrospinal fluid (CFS) decreased from 83.83 ± 4.76 ng mL⁻¹ to 8.66 ± 1.79 ng mL⁻¹ after two months of treatment. Our findings suggest a potential strategy to selectively eliminate senescent cells using chiral nanomaterials and offer a promising strategy for alleviating PD.

The apoptosis pathways of senescent microglia cells induced by chiral NPs under the irradiation of 808 nm laser in the brain of PD mice.     

Dielectrophoretic detection of electrical property changes of stored human red blood cells

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.     

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ialpha. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.     

Inhibitors of the K+(Na+)/H+ exchanger of human red blood cells

The effect of substances as possible inhibitors of the K+(Na+)/H+ exchanger in the human red cell membrane has been tested on the (ouabain+bumetanide+EGTA)-resistant K+ influx in both physiological (HIS) and low ionic strength (LIS) solution with tracer kinetic methods. It is demonstrated that high concentrations of quinacrine (1 mM) and chloroquine (2 mM) inhibit the residual K+ influx in LIS solution to 60% and 85%, respectively, but activate it in HIS solution. Thus, chloroquine suppressed the 10-fold LIS-induced activation of the flux nearly completely. Amiloride derivatives were able to inhibit the K+ influx in both HIS and LIS solution. EIPA (75 microM) reduced the flux by about 20% and 55% in HIS and LIS solution, respectively. Newly developed drugs (HOE 642, 1 mM; HOE 694, 0.5 mM) designed to inhibit Na+/H+ exchanger isoforms showed an inhibition of the residual K+ influx of 40% and 33% in HIS and 65% and 44% in LIS solution, respectively, without haemolysis. The inhibitory effect of HOE 642 persisted in HIS (24%) and LIS (48%) solutions when Cl- was replaced by CH3SO4-. The K(+)-Cl- cotransport inhibitor DIOA (100 microM) stimulated the residual K+ influx in both solutions. It is, therefore, concluded that the K(+)-Cl- cotransporter does not contribute to the residual K+ influx both in HIS and LIS media. Okadaic acid decreased the residual K+ influx by 40% and 25% in HIS and LIS solution, respectively, showing that the residual K+ influx is affected by phosphatases like other ion transport pathways. The results show that the residual K+ influx can be decreased further by inhibiting the K+(Na+)/H+ exchanger. It remains still unclear to what extent the K+(Na+)/H+ exchanger is inhibited by the different substances used. However, the ground state membrane permeability for K+ is much smaller than assumed so far.     

Role of the microfibrillar system in knob action of transformed cells.

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5-4 micron in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15-60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: 1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; 2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; 3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reverse-transformed cells or normal fibroblasts; 4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; 5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.     

溶剂气浮法去除水中的刚果红的研究

以十六烷基三甲基溴化铵为表面活性剂, 与阴离子型染料刚果红形成缔合物, 对该缔合物的溶剂气浮过程进行研究. 研究多种参数对溶剂气浮过程的影响, 如气浮速率、共存溶质的量、pH等参数对溶剂气浮去除率的影响. 研究表明表面活性剂与染料的物质的量之比为2∶1, 约24 min水中刚果红的去除率可达97%;NaCl会大大降低溶剂气浮的去除率;溶剂气浮的速率随着气流速率的增加而增加, 但高速率反而降低溶剂的去除率;共存溶质乙醇存在会使去溶剂去除率降低, 有机溶剂的量对溶剂气浮影响较小;pH中性去除率最佳;考察了不同温度下溶剂气浮的热力学及动力学, 研究表明, 溶剂气浮过程遵从一级动力学, 计算了该过程中的气浮表观活化能为7.48 kJ/mol.     

Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL-CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol-hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL⁻¹ to 6.8 μg mL⁻¹ was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL⁻¹ (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.     

Oxygen dependence of K(+)-Cl- cotransport in human red cell ghosts and sickle cells

KCC activity in normal human red cells (containing haemoglobin A, HbA, and termed HbA cells) is O2-dependent, being active in oxygenated cells but inactive in deoxygenated ones. The mechanism for O2 dependence is unknown but a role for Hb has been suggested. In this paper, we address two main questions. First, do membrane ghosts prepared from HbA cells retain an O2-sensitive KCC activity? Second, how is the response of KCC to changes in O2 tension altered in sickle cell patients heterozygous for HbS and HbC? We found that substantial Cl(-)-dependent K+ influx, indicative of KCC activity, was present in both pink (5-10% normal Hb complement) and white (no measurable Hb) ghosts when equilibrated with air. KCC responded to deoxygenation in pink ghosts only (86 +/- 10% inhibition, mean+/-S.E.M., n = 3), whilst KCC activity in white ghosts remained high (23 +/- 8% inhibition). Results indicate that pink ghosts retain an O2-dependent KCC activity but that this is lost in white ghosts. Second, HbSC-containing red cells showed sickling (88 +/- 3%) when deoxygenated, together with activation of the deoxygenation-induced cation pathway (Psickle) and the Gardos channel. KCC activity, however, was elevated in oxygenated HbSC cells, but inhibited by deoxygenation. Thus Hb polymerisation and sickling could be dissociated from the abnormal response of KCC to deoxygenation observed in HbS-containing red cells. These preparations provide a useful system with which to study the components involved in O2-sensitive membrane transport and why it is perturbed in certain pathological conditions (such as sickle cell disease and oxidant toxicity).     

Determination of cocaine in human urine, plasma and red blood cells by gas-liquid chromatography

This paper describes a sensitive and reliable method for the determination of cocaine in human urine, plasma and red blood cells. Cocaine is extracted into cyclohexane from the biological materials at slightly alkaline pH, reduced with lithium aluminium hydride, acylated with pentafluoropropionic anhydride and detected by an electron capture detector. When compared with a gas chromatography-mass spectrometry method the results of cocaine determination correlated highly (r = 0.986). When cocaine was given intravenously to volunteer subjects only 0.2-1.4% of the administered dose was excreted as unmetabolized cocaine in the first 9 h after administration. Plasma and red blood cell levels of cocaine were also determined by this method after intravenous administration.