RESUMO
INTRODUCTION: The aim of this study was to establish a radio synthesis of (99m)Tc-HYNIC-lactadherin for in vivo studies and to perform biodistribution analysis studies in mice, comparing (99m)Tc-HYNIC-lactadherin to (99m)Tc-HYNIC-annexin V. METHODS: The radiochemical purity of (99m)Tc-HYNIC-lactadherin was optimized by varying the amount of SnCl(2) in the synthesis. Furthermore, the need for bovine serum albumin (BSA) as a stabilizing agent was evaluated by following the stability by radiochemical purity measurement with and without the addition of BSA. A total of 24 mice were assigned to groups of 15 and nine mice, respectively. The animals were sacrificed at different time points; 10 min, 60 min, and 180 min. RESULTS: The synthesis of (99m)Tc-HYNIC-lactadherin for in vivo studies has been optimized to give a stable product without addition of BSA and with a radiochemical purity of more than 95%. Approximately 60% of the injected dose of (99m)Tc-HYNIC-lactadherin was found in the liver and 4-5% could be assigned to kidneys. In contrast, (99m)Tc-HYNIC-annexin V distributes with around 13% and 45% of the injected dose in liver and kidneys, respectively. Over the experimental period (10-180 min) only small distributional changes were observed for both probes. CONCLUSION: In conclusion, the biodistribution of (99m)Tc-HYNIC-lactadherin, a potential new tracer for in vivo quantification of apoptosis, was evaluated. The small renal uptake of (99m)Tc-HYNIC-lactadherin makes it possible to image apoptosis in the kidneys, but the high liver clearance may be a disadvantage during myocardial perfusion.
Assuntos
Apoptose , Proteínas do Leite/farmacocinética , Compostos de Organotecnécio/farmacocinética , Traçadores Radioativos , Animais , Anexina A5/farmacocinética , Apoptose/efeitos dos fármacos , Bovinos , Eletroforese em Gel de Poliacrilamida , Etoposídeo/farmacologia , Células HL-60 , Humanos , Camundongos , Fatores de Tempo , Distribuição Tecidual/efeitos dos fármacosRESUMO
Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical cancer cell line HeLa has been employed as a model system to compare the topographic distribution of PS with the two PS binding proteins as adherent cells enter the apoptotic program. HeLa cells were cultured on glass-bottom Petri dishes, and apoptosis was induced by staurosporine. Fluorescence-labeled lactadherin and/or annexin V were used to detect PS exposure by confocal microscopy. Both lactadherin and annexin V staining revealed PS localized to plasma membrane rim and blebs. In addition, lactadherin identified PS exposure on long filopodia-like extensions, whereas annexin V internalized in granule-like structures. All in all, the data further delineate the differences in PS binding patterns of lactadherin and annexin V.
Assuntos
Apoptose , Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Leite/metabolismo , Fosfatidilserinas/biossíntese , Animais , Anexina A5/metabolismo , Bovinos , Corantes Fluorescentes , Células HeLa , Humanos , Microscopia Confocal , Estaurosporina/farmacologiaRESUMO
BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.