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Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells.

Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins.They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece.

ABSTRACT
Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes. The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

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Related in: MedlinePlus

Increased RBC cytoskeletal protein carbonylation induced by storage in CPDA medium. (A) A representative oxyblot analysis, i.e. immunoblot analysis of cytoskeletons from a donor stained with the anti-DNP antibody. The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kD (right-hand side). (B) Immunoblot of the cytoskeletons probed with anti-human spectrin antibody (internal loading control). (C) The estimation of the oxidative index of the cytoskeletal samples after normalization to control values. The points in the graphs represent the average values and the error bars the standard deviation among the six tested blood donors.
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fig03: Increased RBC cytoskeletal protein carbonylation induced by storage in CPDA medium. (A) A representative oxyblot analysis, i.e. immunoblot analysis of cytoskeletons from a donor stained with the anti-DNP antibody. The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kD (right-hand side). (B) Immunoblot of the cytoskeletons probed with anti-human spectrin antibody (internal loading control). (C) The estimation of the oxidative index of the cytoskeletal samples after normalization to control values. The points in the graphs represent the average values and the error bars the standard deviation among the six tested blood donors.

Mentions: The production of carbonyl groups (aldehydes and ketones) on protein side chains (especially of Pro, Arg, Lys and Thr) is a common phenomenon of protein oxidation. The protein carbonyl groups were presently used as biomarkers of protein oxidative stress in order to clarify whether the storage affects the oxidative status of cytoskeletal proteins as well. As shown in Fig. 3, the oxidative index of the cytoskeletal proteins, namely the ratio of the total oxyblot signal (Fig. 3A), either to the immunoblotting signal of a reference protein (like spectrin) (Fig. 3B), or to the sum of the immunoblotted proteins that were revealed after the Red Ponceau staining of each sample (data not shown), was found increased after 10 days of RBC storage, which signalized oxidative modifications of spectrin and possibly other cytoskeletal proteins. The higher levels of cytoskeletal protein carbonyl groups were observed after 35 days of storage in CPDA (Fig. 3C). “Improved” amounts of cytoskeleton-bound Hb (Fig. 2B) and protein carbonylation levels (Fig. 3C) were observed in outdated samples (day 43).


Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells.

Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS - J. Cell. Mol. Med. (2007 Jan-Feb)

Increased RBC cytoskeletal protein carbonylation induced by storage in CPDA medium. (A) A representative oxyblot analysis, i.e. immunoblot analysis of cytoskeletons from a donor stained with the anti-DNP antibody. The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kD (right-hand side). (B) Immunoblot of the cytoskeletons probed with anti-human spectrin antibody (internal loading control). (C) The estimation of the oxidative index of the cytoskeletal samples after normalization to control values. The points in the graphs represent the average values and the error bars the standard deviation among the six tested blood donors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401228&req=5

fig03: Increased RBC cytoskeletal protein carbonylation induced by storage in CPDA medium. (A) A representative oxyblot analysis, i.e. immunoblot analysis of cytoskeletons from a donor stained with the anti-DNP antibody. The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kD (right-hand side). (B) Immunoblot of the cytoskeletons probed with anti-human spectrin antibody (internal loading control). (C) The estimation of the oxidative index of the cytoskeletal samples after normalization to control values. The points in the graphs represent the average values and the error bars the standard deviation among the six tested blood donors.
Mentions: The production of carbonyl groups (aldehydes and ketones) on protein side chains (especially of Pro, Arg, Lys and Thr) is a common phenomenon of protein oxidation. The protein carbonyl groups were presently used as biomarkers of protein oxidative stress in order to clarify whether the storage affects the oxidative status of cytoskeletal proteins as well. As shown in Fig. 3, the oxidative index of the cytoskeletal proteins, namely the ratio of the total oxyblot signal (Fig. 3A), either to the immunoblotting signal of a reference protein (like spectrin) (Fig. 3B), or to the sum of the immunoblotted proteins that were revealed after the Red Ponceau staining of each sample (data not shown), was found increased after 10 days of RBC storage, which signalized oxidative modifications of spectrin and possibly other cytoskeletal proteins. The higher levels of cytoskeletal protein carbonyl groups were observed after 35 days of storage in CPDA (Fig. 3C). “Improved” amounts of cytoskeleton-bound Hb (Fig. 2B) and protein carbonylation levels (Fig. 3C) were observed in outdated samples (day 43).

Bottom Line: A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins.They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece.

ABSTRACT
Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes. The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

Show MeSH
Related in: MedlinePlus