Spectrally and Time-Resolved Fluorescence Imaging of 22-NBD-Cholesterol in Human Peripheral Blood Mononuclear Cells in Chronic Kidney Disease Patients.

The interaction of the fluorescent probe 22-NBD-cholesterol with membranes of human peripheral blood mononuclear cells (PBMC) was tested by time- and spectrally resolved fluorescence imaging to monitor the disturbance of lipid metabolism in chronic kidney disease (CKD) and its treatment with statins. Blood samples from healthy volunteers (HV) and CKD patients, either treated or untreated with statins, were compared. Spectral imaging was done using confocal microscopy at 16 spectral channels in response to 458 nm excitation. Time-resolved imaging was achieved by time-correlated single photon counting (TCSPC) following excitation at 475 nm. The fluorescence of 22-NBD-cholesterol was mostly integrated into plasmatic membrane and/or intracellular membrane but was missing from the nuclear region. The presence of two distinct spectral forms of 22-NBD-cholesterol was uncovered, with significant variations between studied groups. In addition, two fluorescence lifetime components were unmasked, changing in CKD patients treated with statins. The gathered results indicate that 22-NBD-cholesterol may serve as a tool to study changes in the lipid metabolism of patients with CKD to monitor the effect of statin treatment.

Analysis of fluorescent ceramide and sphingomyelin analogs: a novel approach for in vivo monitoring of sphingomyelin synthase activity.

A novel sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method was developed for real-time monitoring of relative sphingomyelin synthase (SMS) activity based on the measurement of a fluorescent ceramide (Cer) analog and its metabolite, a fluorescent sphingomyelin (CerPCho) analog, in plasma. Analyses were conducted using HPLC-FLD following a protein precipitation procedure. The chromatographic separations were carried out on an Agilent C18 RP column (150 × 4.6 mm, 5 µm) based on a methanol-0.1 % trifluoroacetic acid aqueous solution (88:12, by vol) elution at a flow-rate of 1 mL/min. The limit of quantification in plasma was 0.05 µM for both the fluorescent Cer analog and its metabolite. Significant differences in the fluorescent Cer analog and its metabolite concentration ratio at 5 min were found between vehicle control group and three D2 (a novel SMS inhibitor) dose groups (P < 0.05). Dose-dependent effects (D2 doses: 0, 2.5, 5, 10 mg/kg) were observed. Our method could be used to detect relative SMS activity in biochemical assays and to screen potential SMS inhibitors in vivo. D2 was found to be a potent SMS inhibitor in vivo, and may have a potential antiatherosclerotic effect, which is under further study. D609 was also selected as another model SMS inhibitor to validate our newly developed method.

Nonextractive procedure and precolumn derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for trace determination of trimetazidine in plasma by high-performance liquid chromatography with fluorescence detection.

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed and validated in a single laboratory for the trace determination of trimetazidine (TMZ) in human plasma. Fluoxetine (FLX) was used as the internal standard. TMZ and FLX were isolated from plasma by protein precipitation with acetonitrile and derivatized by heating with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole in pH 8 borate buffer at 70 degrees C for 30 min. Separations were performed in the isocratic mode on a Nucleosil CN column with the mobile phase acetonitrile-10 mM sodium acetate buffer (pH 3.5)-methanol (47 + 47 + 6, v/v/v) at a flow rate of 1.0 mL/min. The derivatized samples were excited at 470 nm and monitored at an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with a good correlation coefficient (r = 0.9997, n = 5) was obtained for the peak area ratio of TMZ to FLX and for TMZ concentrations of 1-120 ng/mL. The proposed method has the lowest limits of detection and quantitation reported to date for the determination of TMZ in plasma with values of 0.3 and 0.95 ng/mL, respectively. The values for intra- and interassay precision were satisfactory; the relative standard deviations were < or =4.04%. The accuracy of the method was demonstrated; the recoveries of TMZ from spiked human plasma were 98.13-102.83 +/- 0.2-4.04%. The method has high throughput because of its simple sample preparation procedure and short run time (<10 min). The results demonstrated that the proposed method would have great value when applied in pharmacokinetic studies for TMZ.

Determination of lisinopril in dosage forms and spiked human plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) followed by spectrophotometry or HPLC with fluorimetric detection.

Two sensitive, simple and specific methods based on spectrophotometry and reversed-phase HPLC with fluorimetric detection are described for the determination of lisinopril in dosage forms as well as in spiked human plasma using solid phase extraction (SPE) procedures. Both methods are based on the derivatization of lisinopril with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 9 to yield a yellow, fluorescent product. The spectrophotometric method depends on measuring the formed yellow color at 470 nm after optimization of the reaction conditions. The HPLC method is based on measurement of the derivatized product using fluorescence detection at 540 nm (excitation at 470 nm). The separation of the derivatized drug, the excess reagent and the internal standard (bumetanide) was performed on a reversed-phase ODS column using isocratic elution with methanol-0.02 M sodium dihydrogen phosphate, pH 3.0 (55:45, v/v) at a flow rate of 1.0 ml/min. The calibration graphs were linear over the concentration ranges 2-20 or 0.02-3.2 microg/ml of lisinopril with minimum detectability of 0.3 and 0.008 microg/ml (6.1 x 10(-7) and 1.7 x 10(-8)M) for the spectrophotometric and the HPLC methods, respectively. The proposed methods were applied without any interference from the tablet excipients for the determination of lisinopril in dosage forms, either alone or co-formulated with hydrochlorothiazide. Furthermore, the use of the HPLC method was extended to the in vitro determination of the drug in spiked human plasma. Interference from endogenous amino acids has been overcomed by using the solid phase extraction technique, the percentage recovery (n=6) was 101.6+/-3.35.

Comparison between Ca2+-induced scrambling of various fluorescently labelled lipid analogues in red blood cells.

Treatment of red blood cells with calcium and ionomycin causes activation of the lipid scramblase, a putative membrane protein catalysing flip-flop of (phospho)lipids. Various fluorescent 1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl (C(6)-NBD) analogues were tested for transbilayer movement across the plasma membrane of red blood cells. Among these phospholipid analogues were phosphatidylgalactose, phosphatidylmaltose and phosphatidylmaltotriose, which were obtained from C(6)-NBD-phosphatidylcholine by phospholipase D-catalysed transphosphatidylation. The inward movement after the onset of scrambling was monitored by extraction of the non-internalized probe with BSA. We demonstrate that both the amino group and the size of the headgroup determine the kinetics of lipid scrambling, and that lipids with a ceramide backbone migrate much more slowly than glycerophospholipids with the same headgroup.

Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1).

The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.

Pathways for flip-flop of mono- and di-anionic phospholipids in the erythrocyte membrane.

The inward translocations (flip), from the outer to the inner membrane leaflet of human erythrocytes, of di-anionic NBD-labeled phospholipids containing as a head group phosphate esters of glycolate, butyrate and hydroxyethanesulfonate are slow processes (k = 0.005-0.008 h-1, 37 degrees C) at pH 7.4. A decrease of pH highly stimulates the flip. A major role of the anion exchanger (AE1), band 3, in this flip is indicated by (a) the strong inhibition of the flip (55-85%) by stilbene disulfonates and other inhibitors of anion transport, (b) the stimulation and loss of pH dependence of the flip after modification of band 3 by Woodward's reagent K and NaBH4, and (c) the stimulation of the flip after proteolytic cleavage of band 3 by papain. The flip of mono-anionic NBD-phospholipids with phosphate esters of glycerol, glycol, methanol, butanol and benzyl alcohol is much faster than that of their dianionic analogs (k = 0.04 to > 3.0 h-1, 37 degrees C). It is inhibited by stilbene disulfonates to a decreasing extent (35 to 0%) and is not affected by several reversible inhibitors of anion exchange. This indicates a minor component of band-3-mediated flip and a major component of nonmediated flip. The outward translocations (flop), from the inner to outer membrane leaflet, of both mono- and di-anionic phospholipids are very fast (1.0-5.9 h-1), ATP-dependent and inhibitable by vanadate, fluoride, SH-reagents or Mg(2+)-depletion of cells and thereby likely to be largely mediated by a 'floppase'. The stationary distributions of the NBD-labeled anionic phospholipids are asymmetric to an extent (outer to inner leaflet ratio 2-9) correlating with the ratio of the rates of the outward and the inward translocation. Thus, asymmetry is largely abolished by blockage of the floppase-mediated translocation.

Cholesterol redistribution within human platelet plasma membrane: evidence for a stimulus-dependent event.

The fluorescent analog NBD-phosphatidylethanolamine and the analogs of cholesterol NBD-cholesterol and cholestatrienol were used to study the distribution of these lipids within the plasma membrane bilayer of human platelets. The probes were incorporated into platelets using phosphatidylcholine donor vesicles. The distribution of NBD lipid and of cholestatrienol in the platelet plasma membrane bilayer was followed by quenching with dithionite and TNBS, respectively. The t1/2 of cholestatrienol incorporation into platelet membranes was 39 min, and approximately 65% of the probe was quenched by addition of TNBS. When platelets were exposed to collagen or to ADP, a portion of the probe became inaccessible to quenching. Within 2 min of stimulation by collagen (10 micrograms/mL), the percentage of cholestatrienol fluorescence quenched by TNBS decreased to 45%. The fluorescent probe was not found to be associated either with the intracellular membranes or in the extracellular media after collagen stimulation. Similar data were obtained with NBD-cholesterol, but the decrease in accessibility of this probe to quenching was considerably slower. The redistribution of endogenous membrane cholesterol was also measured using cholesterol oxidase. Exposure of platelets to collagen decreased the accessibility of endogenous membrane cholesterol to enzymatic oxidation with cholesterol oxidase. Taken together, the foregoing observations are consistent with the stimulus-dependent translocation of cholesterol out of the outer monolayer. Coincident with the redistribution of cholesterol is the reciprocal movement of NBD-phosphatidylethanolamine into the outer monolayer. In the presence of the chaotropic agents urea and guanidine HCl, the movement of cholestatrienol upon collagen stimulation was prevented, but the redistribution of NBD-phosphatidylethanolamine was still detected. We propose that cholesterol translocates to the inner platelet monolayer following collagen stimulation, but the possibility that the sterol moves laterally within the outer membrane monolayer cannot be rigorously excluded.

Transverse redistribution of phospholipids during human platelet activation: evidence for a vectorial outflux specific to aminophospholipids.

The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.

Polyamine inhibition of transbilayer movement of plasma membrane phospholipids in the erythrocyte ghost.

The resting plasma membrane of circulating blood cells demonstrates an asymmetric distribution of the phospholipid classes across the bilayer that is altered during cellular activation. To better understand the mechanisms governing transbilayer distribution of phospholipids, studies were conducted using the erythrocyte ghost, in which plasma membrane leaflet distribution of phospholipids can be readily probed. Preparation of ghosts by hypotonic lysis at increasingly high dilution markedly enhanced (up to 10-fold) calcium-induced (50-500 microM) transbilayer movement of phospholipids. The enhanced transbilayer movement was assessed by translocation of exogenously added sn-2-[6[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-labeled phosphatidylcholine and phosphatidylserine from the plasma membrane outer leaflet to the inner leaflet and vice versa, as well as transbilayer movement of endogenous phosphatidylserine to the outer leaflet. It was found that phospholipid movement was bidirectional and also nonspecific with regard to polar head group. It was further demonstrated that preparation of ghosts at increasing dilution resulted in depletion of cellular polyamines and that physiologic replenishment of spermine, and to a lesser extent spermidine, resulted in significant inhibition (50 and 25%, respectively) of transbilayer movement of phospholipids. Replenishment of other di- and polyamines demonstrated that inhibition was not simply dependent on total cationic charge but rather on charge density and suggestive of specific interaction of the polyamines, particularly spermine, with the plasma membrane. As most cells demonstrate both a high degree of regulation in maintenance of polyamine levels and the means for facile shifts within cellular polyamine pools, it is suggested that loss of membrane asymmetry during cellular activation may be mediated in part through enhanced transbilayer movement of phospholipids due to altered polyamine-membrane associations.

Regional differences in glucose transport in the mouse hippocampus.

In order to observe glucose transport into the brain, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG), a non-metabolizable and fluorescent glucose analogue, was injected intravenously into mice. After ascertaining that this glucose analogue is non-metabolizable in the brain, the NBDG contents in the blood and brain were measured quantitatively by spectrofluorimetry at 0, 0.5, 2, 5, 10 and 30 min after intravenous injection. The NBDG content in the blood decreased markedly with time, whereas in the brain it rapidly decreased, then gradually increased after 2 min. Glucose transport into the hippocampus was observed with a confocal laser scanning microscope. At 0.5 min, NBGD was seen to be highly concentrated on the vascular wall. Using the confocal mode, it was found that the fluorescence was unevenly distributed on the microvessel wall, suggesting local differences of glucose transport in the vascular wall. At 5 min, the fluoresent intensity of the vascular wall was markedly decreased, whereas relatively intense fluorescence was observed in the cerebral parenchyma of the stratum lacunosum-moleculare and stratum pyramidale of Ca3. At 10 min, a weak fluoresence was diffusely distributed in the hippocampus. As to the localization of NBDG in the brain, capillary endothelium (luminal and abluminal membrane), basement membrane, and the feet of the astrocytes are discussed.

Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells.

Human red blood cells (RBC) were separated by density on self-forming Percoll gradients into five distinct populations. The transbilayer movement and equilibrium distribution of 1-oleoyl-2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminocaproyl)phosphatidylserine (NBD-PS) was slower in dense cells and equilibrated in the inner leaflet of these cells to a lesser degree than in light cells. Conversely, the outward movement of the lipid was slower in light cells. Assessment of endogenous PS in the cells' outer leaflet by the prothrombinase activity of externalized PS revealed an increase in its presence at the cell surface with increasing cell density. The presence of PS on the cell surface directly correlated with the propensity of the RBC to bound by autologous monocytes. To determine whether increased cell density is associated with increased cell age, the in vivo clearance of density-separated murine RBC was monitored in syngeneic mice. The T1/2 of circulation of light cells was about twice that of dense cells. The majority of the cleared cells localized in the spleen. Studies carried out in antibody-deficient SCID mice indicated that RBC were cleared via a mechanism that was independent of antibody. These data suggest that cell age is related to cell density and that cells of increasing age and density display higher amounts of PS in their outer leaflet.